Anti-pro792 antibodies

ABSTRACT

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

This application is a continuation of, and claims priority under 35 USC§120 to U.S. application Ser. No. 09/918,585, filed Jul. 30, 2001, nowabandoned which is a continuation of, and claims priority under 35 USC§120 to PCT Application PCT/US00/04341, filed Feb. 18, 2000, whichclaims priority under 35 USC §119 to U.S. Provisional Application60/131,445, filed Apr. 28, 1999, and under 35 USC §120, as acontinuation-in-part, of PCT Application PCT/US99/28313, filed Nov. 30,1999, which is a continuation-in-part of, and claims priority under 35USC §120 to U.S. application Ser. No. 09/380,138, filed Aug. 25, 1999,now abandoned, which is the National Stage Application filed under 35USC §371 of PCT Application PCT/US99/05028, filed Mar. 8, 1999, whichclaims priority under 35 USC §119 to U.S. Provisional Application60/084,637, filed May 17, 1998.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides encoded by that DNA.

BACKGROUND OF THE INVENTION

Extracellular proteins play an important role in the formation,differentiation and maintenance of multicellular organisms. The fate ofmany individual cells, e.g., proliferation, migration, differentiation,or interaction with other cells, is typically governed by informationreceived from other cells and/or the immediate environment. Thisinformation is often transmitted by secreted polypeptides (for instance,mitogenic factors, survival factors, cytotoxic factors, differentiationfactors, neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Thesesecreted polypeptides or signaling molecules normally pass through thecellular secretory pathway to reach their site of action in theextracellular environment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play an important role in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. Such membrane-bound proteins and cellreceptors include, but are not limited to, cytokine receptors, receptorkinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction. Effortsare being undertaken by both industry and academia to identify new,native receptor proteins. Many efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel receptor proteins.

We herein describe the identification and characterization of novelsecreted and transmembrane polypeptides and novel nucleic acids encodingthose polypeptides.

1. PRO213

Human growth arrest-specific gene 6 (gas6) encodes a protein that isexpressed in a variety of different tissues and which has been reportedto be highly expressed during periods of serum starvation and negativelyregulated during growth induction. See Manfioletti et al., Mol. Cell.Biol. 13(8):4976–4985 (1993) and Stitt et al., Cell 80:661–670 (1995).Manfioletti et al. (1993), supra, have suggested that the gas6 proteinis member of the vitamin dependent family of proteins, wherein themembers of the latter family of proteins (which include, for example,Protein S, Protein C and Factor X) all play regulatory roles in theblood coagulation pathway. Thus, it has been suggested that gas6 mayplay a role in the regulation of a protease cascade relevant in growthregulation or in the blood coagulation cascade.

Given the physiological importance of the gas6 protein, efforts arecurrently being undertaken by both industry and academia to identifynew, native proteins which are homologous to gas6. Many of these effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins, specifically those having homology to gas6. Examplesof such screening methods and techniques are described in the literature[see, for example, Klein et al., Proc. Natl. Acad. Sci., 93:7108–7113(1996); U.S. Pat. No. 5,536,637)]. We herein describe the identificationof a novel polypeptide which has homology to the gas6 polypeptide.

2. PRO274

The 7-transmembrane (“7TM”) proteins or receptors, also referred to inthe literature as G-protein coupled receptors, are specialized proteinsdesigned for recognition of ligands and the subsequent signaltransduction of information contained within those ligands to themachinery of the cell. The primary purpose of cell surface receptors isto discriminate appropriate ligands from the various extracellularstimuli which each cell encounters, then to activate an effector systemthat produces an intracellular signal, thereby controlling cellularprocesses. [Dohlman, H., Ann. Rev. Biochem., 60:653 (1991)]. The abilityof 7TM receptors to bind ligand to a recognition domain andallosterically transmit the information to an intracellular domain is aspecialized feature of 7TM proteins [Kenakin, T., Pharmacol. Rev. 48:43(1996)]. The gene family which encodes the 7TM receptors or G-proteinlinked receptors encode receptors which recognize a large number ofligands, including but not limited to, C5a, interleukin 8 and relatedchemokines. Research in this area suggests that distinct signals at thecell surface feed into common pathways of cell activation. [Gerard, C.and Gerard, N., Curr. Op. Immunol., 6:140 (1994), Gerard, C. and Gerard,N., Ann. Rev. Immunol., 12:775 (1994)]. The superfamily of 7TM orG-protein coupled receptors contains several hundred members able torecognize various messages such as photons, ions and amino acids amongothers [Schwartz, T. W., et al., H., Trends in Pharmacol. Sci.,17(6):213 (1996)].

[Dohman, H., Ann. Rev. Biochem. 60:653 (1991)]. [Schwartz, T. W., etal., H., Eur. J. Pharm. Sci., 2:85 (1994)]. We describe herein theidentification of a novel polypeptide (designated herein as PRO274)which has homology to the 7 transmembrane segment receptor proteins andthe Fn54 protein.3. PRO300

The Diff 33 protein is over-expressed in mouse testicular tumors. Atpresent its role is unclear, however, it may play a role in cancer.Given the medical importance of understanding the physiology of cancer,efforts are currently being under taken to identify new, native proteinswhich are involved in cancer. We describe herein the identification of anovel polypeptide which has homology to Diff 33, designated herein asPRO300.

4. PRO284

Efforts are currently being undertaken to identify and characterizenovel transmembrane proteins. We herein describe the identification andcharacterization of a novel transmembrane polypeptide, designated hereinas PRO284.

5. PRO296

Cancerous cells often express numerous proteins that are not expressedin the corresponding normal cell type or are expressed at differentlevels than in the corresponding normal cell type. Many of theseproteins are involved in inducing the transformation from a normal cellto a cancerous cell or in maintaining the cancer phenotype. As such,there is significant interest in identifying and characterizing proteinsthat are expressed in cancerous cells. We herein describe theidentification and characterization of a novel polypeptide havinghomology to the sarcoma-amplified protein SAS, designated herein asPRO296.

6. PRO329

Immunoglobulin molecules play roles in many important mammalianphysiological processes. The structure of immunoglobulin molecules hasbeen extensively studied and it has been well documented that intactimmunoglobulins possess distinct domains, one of which is the constantdomain or F_(c) region of the immunoglobulin molecule. The F_(c) domainof an immunoglobulin, while not being directly involved in antigenrecognition and binding, does mediate the ability of the immunoglobulinmolecule, either uncomplexed or complexed with its respective antigen,to bind to F_(c) receptors either circulating in the serum or on thesurface of cells. The ability of an F_(c) domain of an immunoglobulin tobind to an F_(c) receptor molecule results in a variety of importantactivities, including for example, in mounting an immune responseagainst unwanted foreign particles. As such, there is substantialinterest in identifying novel F_(c) receptor proteins and subunitsthereof. We herein describe the identification and characterization of anovel polypeptide having homology to a high affinity immunoglobulinF_(c) receptor protein, designated herein as PRO329.

7. PRO362

Colorectal carcinoma is a malignant neoplastic disease which has a highincidence in the Western world, particularly in the United States.Tumors of this type often metastasize through lymphatic and vascularchannels and result in the death of some 62,000 persons in the UnitedStates annually.

Monoclonal antibody A33 (mAbA33) is a murine immunoglobulin that hasundergone extensive preclinical analysis and localization studies inpatients inflicted with colorectal carcinoma (Welt et al., J. Clin.Oncol. 8:1894–1906 (1990) and Welt et al., J. Clin. Oncol. 12:1561–1571(1994)). mAbA33 has been shown to bind to an antigen found in and on thesurface of normal colon cells and colon cancer cells. In carcinomasoriginating from the colonic mucosa, the A33 antigen is expressedhomogeneously in more than 95% of the cases. The A33 antigen, however,has not been detecting in a wide range of other normal tissues, i.e.,its expression appears to be rather organ specific. Therefore, the A33antigen appears to play an important role in the induction of colorectalcancer.

Given the obvious importance of the A33 antigen in tumor cell formationand/or proliferation, there is substantial interest in identifyinghomologs of the A33 antigen. In this regard, we herein describe theidentification and characterization of a novel polypeptide havinghomology to the A33 antigen protein, designated herein as PRO362.

8. PRO363

The cell surface protein HCAR is a membrane-bound protein that acts as areceptor for subgroup C of the adenoviruses and subgroup B of thecoxsackieviruses. Thus, HCAR may provide a means for mediating viralinfection of cells in that the presence of the HCAR receptor on thecellular surface provides a binding site for viral particles, therebyfacilitating viral infection.

In light of the physiological importance of membrane-bound proteins andspecifically those which serve a cell surface receptor for viruses,efforts are currently being undertaken by both industry and academia toidentify new, native membrane-bound reeptor proteins. Many of theseefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel receptor proteins.We herein describe a novel membrane-bound polypeptide having homology tothe cell surface protein HCAR and to various tumor antigens includingA33 and carcinoembryonic antigen, designated herein as PRO363, whereinthis polypeptide may be a novel cell surface virus receptor or tumorantigen.

9. PRO868

Control of cell numbers in mammals is believed to be determined, inpart, by a balance between cell proliferation and cell death. One formof cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al.,Bio/Technology, 12:487–493 (1994); Steller et al., Science,267:1445–1449 (1995)]. Apoptotic cell death naturally occurs in manyphysiological processes, including embryonic development and clonalselection in the immune system [Itoh et al., Cell, 66:233–243 (1991)].Decreased levels of apoptotic cell death have been associated with avariety of pathological conditions, including cancer, lupus, and herpesvirus infection [Thompson, Science, 267:1456–1462 (1995)]. Increasedlevels of apoptotic cell death may be associated with a variety of otherpathological conditions, including AIDS, Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,retinitis pigmentosa, cerebellar degeneration, a plastic anemia,myocardial infarction, stroke, reperfusion injury, and toxin-inducedliver disease [see, Thompson, supra].

Apoptotic cell death is typically accompanied by one or morecharacteristic morphological and biochemical changes in cells, such ascondensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss ofmitochondrial function. A variety of extrinsic and intrinsic signals arebelieved to trigger or induce such morphological and biochemicalcellular changes [Raff, Nature, 356:397–400 (1992); Steller, supra;Sachs et al., Blood, 82:15 (1993)]. For instance, they can be triggeredby hormonal stimuli, such as glucocorticoid hormones for immaturethymocytes, as well as withdrawal of certain growth factors[Watanabe-Fukunaga et al., Nature 356:314–317 (1992)]. Also, someidentified oncogenes such as myc, rel, and E1A, and tumor suppressors,like p53, have been reported to have a role in inducing apoptosis.Certain chemotherapy drugs and some forms of radiation have likewisebeen observed to have apoptosis-inducing activity [Thompson, supra].

Various molecules, such as tumor necrosis factor-α (“TNF-α”), tumornecrosis factorβ (“TNF-β” or “lymphotoxin-α”), lymphotoxin-β (“LT-β”),CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1ligand (also referred to as Fas ligand or CD95 ligand), and Apo-2 ligand(also referred to as TRAIL) have been identified as members of the tumornecrosis factor (“TNF”) family of cytokines [See, e.g., Gruss and Dower,Blood, 85:3378–3404 (1995); Pitti et al., J. Biol. Chem.,271:12687–12690 (1996); Wiley et al., Immunity, 3.673–682 (1995);Browning et al., Cell, 72:847–856 (1993); Armitage et al. Nature,357:80–82 (1992), WO 97/01633 published Jan. 16, 1997; WO 97/25428published Jul. 17, 1997]. Among these molecules, TNF-α, TNF-β, CD30ligand, 4-1BB ligand, Apo-1 ligand, and Apo-2 ligand (TRAIL) have beenreported to be involved in apoptotic cell death. Both TNF-α and TNF-βhave been reported to induce apoptotic death in susceptible tumor cells[Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al.,Eur. J. Immunol., 17:689 (1987)]. Zheng et al. have reported that TNF-αis involved in post-stimulation apoptosis of CD8-positive T cells [Zhenget al., Nature, 377:348–351 (1995)]. Other investigators have reportedthat CD30 ligand may be involved in deletion of self-reactive T cells inthe thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium onProgrammed Cell Death, Abstr. No. 10, (1995)].

Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called lprand gld, respectively) have been associated with some autoimmunedisorders, indicating that Apo-1 ligand may play a role in regulatingthe clonal deletion of self-reactive lymphocytes in the periphery[Krammer et al., Curr. Op. Immunol., 6:279–289 (1994); Nagata et al.,Science, 267:1449–1456 (1995)]. Apo-1 ligand is also reported to inducepost-stimulation apoptosis in CD4-positive T lymphocytes and in Blymphocytes, and may be involved in the elimination of activatedlymphocytes when their function is no longer needed [Krammer et al.,supra; Nagata et al., supra]. Agonist mouse monoclonal antibodiesspecifically binding to the Apo-1 receptor have been reported to exhibitcell killing activity that is comparable to or similar to that of TNF-α[Yonehara et al., J. Exp. Med., 169:1747–1756 (1989)].

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1)and 75-kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem.,264:14927–14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci.,87:3127–3131 (1990); EP 417,563, published Mar. 20, 1991] and human andmouse cDNAs corresponding to both receptor types have been isolated andcharacterized [Loetscher et al., Cell, 61:351 (1990); Schall et al.,Cell, 61:361 (1990); Smith et al., Science, 248:1019–1023 (1990); Lewiset al., Proc. Natl. Acad. Sci., 88:2830–2834 (1991); Goodwin et al.,Mol. Cell. Biol., 11:3020–3026 (1991)]. Extensive polymorphisms havebeen associated with both TNF receptor genes [see, e.g., Takao et al.,Immunogenetics, 37:199–203 (1993)]. Both TNFRs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors are found naturally also as soluble TNF-binding proteins[Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A., 87:8331 (1990)]. More recently, the cloning ofrecombinant soluble TNF receptors was reported by Hale et al. [J. Cell.Biochem. Supplement 15SF, 1991, p. 113 (P424)].

The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2)contains a repetitive amino acid sequence pattern of four cysteine-richdomains (CRDs) designated 1 through 4, staring from the NH₂—terminus.Each CRD is about 40 amino acids long and contains 4 to 6 cysteineresidues at positions which are well conserved [Schall et al., supra;Loetscher et al., supra; Smith et al., supra; Nophar et al., supra;Kohno et al., supra]. In TNFR1, the approximate boundaries of the fourCRDs are as follows: CRD1-amino acids 14 to about 53; CRD2-amino acidsfrom about 54 to about 97; CRD3-amino acids from about 98 to about 138;CRD4-amino acids from about 139 to about 167. In TNFR2, CRD1 includesamino acids 17 to about 54; CRD2-amino acids from about 55 to about 97;CRD3-amino acids from about 98 to about 140; and CRD4-amino acids fromabout 141 to about 179 [Banner et al., Cell, 73:431–435 (1993)]. Thepotential role of the CRDs in ligand binding is also described by Banneret al., supra.

A similar repetitive pattern of CRDs exists in several othercell-surface proteins, including the p75 nerve growth factor receptor(NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J.,8:1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J., 9:1063(1990)] and the Fas antigen [Yonehara et al., supra and Itoh et al.,Cell, 66:233–243 (1991)]. CRDs are also found in the soluble TNFR(sTNFR)-like T2 proteins of the Shope and myxoma poxviruses [Upton etal., Virology, 160:20–29 (1987); Smith et al., Biochem. Biophys. Res.Commun., 176:335 (1991); Upton et al., Virology, 184:370 (1991)].Optimal alignment of these sequences indicates that the positions of thecysteine residues are well conserved. These receptors are sometimescollectively referred to as members of the TNF/NGF receptor superfamily.Recent studies on p75NGFR showed that the deletion of CRD1 [Welcher, A.A. et al., Proc. Natl. Acad. Sci. USA, 88:159–163 (1991)] or a 5-aminoacid insertion in this domain [Yan, H. and Chao, M. V., J. Biol. Chem.,266:12099–12104 (1991)] had little or no effect on NGF binding [Yan, H.and Chao, M. V., supra]. p75 NGFR contains a proline-rich stretch ofabout 60 amino acids, between its CRD4 and transmembrane region, whichis not involved in NGF binding [Peetre, C. et al., Eur. J. Hematol.,41:414–419 (1988); Seckinger, P. et al., J. Biol. Chem., 264:11966–11973(1989); Yan, H. and Chao, M. V., supra]. A similar proline-rich regionis found in TNFR2 but not in TNFR1.

The TNF family ligands identified to date, with the exception oflymphotoxin-α, are type II transmembrane proteins, whose C-terminus isextracellular. In contrast, most receptors in the TNF receptor (TNFR)family identified to date are type I transmembrane proteins. In both theTNF ligand and receptor families, however, homology identified betweenfamily members has been found mainly in the extracellular domain(“ECD”). Several of the TNF family cytokines, including TNF-α, Apo-1ligand and CD40 ligand, are cleaved proteolytically at the cell surface;the resulting protein in each case typically forms a homotrimericmolecule that functions as a soluble cytokine. TNF receptor familyproteins are also usually cleaved proteolytically to release solublereceptor ECDs that can function as inhibitors of the cognate cytokines.

Recently, other members of the TNFR family have been identified. Suchnewly identified members of the TNFR family include CAR1, HVEM andosteoprotegerin (OPG) [Brojatsch et al., Cell, 87:845–855 (1996);Montgomery et al., Cell, 87:427–436 (1996); Marsters et al., J. Biol.Chem., 272:14029–14032 (1997); Simonet et al., Cell, 89:309–319 (1997)].Unlike other known TNFR-like molecules, Simonet et al., supra, reportthat OPG contains no hydrophobic transmembrane-spanning sequence.

Moreover, a new member of the TNF/NGF receptor family has beenidentified in mouse, a receptor referred to as “GITR” for“glucocorticoid-induced tumor necrosis factor receptor family-relatedgene” [Nocentini et al., Proc. Natl. Acad. Sci. USA 94:6216–6221(1997)]. The mouse GITR receptor is a 228 amino acid type Itransmembrane protein that is expressed in normal mouse T lymphocytesfrom thymus, spleen and lymph nodes. Expression of the mouse GITRreceptor was induced in T lymphocytes upon activation with anti-CD3antibodies, Con A or phorbol 12-myristate 13-acetate. It was speculatedby the authors that the mouse GITR receptor was involved in theregulation of T cell receptor-mediated cell death.

In Marsters et al., Curr. Biol., 6:750 (1996), investigators describe afull length native sequence human polypeptide, called Apo-3, whichexhibits similarity to the TNFR family in its extracellularcysteine-rich repeats and resembles TNFR1 and CD95 in that it contains acytoplasmic death domain sequence [see also Marsters et al., Curr.Biol., 6:1669 (1996)]. Apo-3 has also been referred to by otherinvestigators as DR3, ws1-1 and TRAMP [Chinnaiyan et al., Science,274:990 (1996); Kitson et al., Nature, 384:372 (1996); Bodmer et al.,Immunity, 6:79 (1997)].

Pan et al. have disclosed another TNF receptor family member referred toas “DR4” [Pan et al., Science, 276:111–113 (1997)]. The DR4 was reportedto contain a cytoplasmic death domain capable of engaging the cellsuicide apparatus. Pan et al. disclose that DR4 is believed to be areceptor for the ligand known as Apo-2 ligand or TRAIL.

In Sheridan et al., Science, 277:818–821 (1997) and Pan et al., Science,277:815–818 (1997), another molecule believed to be a receptor for theApo-2 ligand (TRAIL) is described. That molecule is referred to as DR5(it has also been alternatively referred to as Apo-2). Like DR4, DR5 isreported to contain a cytoplasmic death domain and be capable ofsignaling apoptosis.

In Sheridan et al., supra, a receptor called DcR1 (or alternatively,Apo-2DcR) is disclosed as being a potential decoy receptor for Apo-2ligand (TRAIL). Sheridan et al. report that DcR1 can inhibit Apo-2ligand function in vitro. See also, Pan et al., supra, for disclosure onthe decoy receptor referred to as TRID.

For a review of the TNF family of cytokines and their receptors, seeGruss and Dower, supra.

As presently understood, the cell death program contains at least threeimportant elements—activators, inhibitors, and effectors; in C. elegans,these elements are encoded respectively by three genes, Ced4, Ced-9 andCed-3 [Steller, Science, 267:1445 (1995); Chinnaiyan et al., Science,275:1122–1126 (1997); Wang et al., Cell, 90:1–20 (1997)]. Two of theTNFR family members, TNFR1 and Fas/Apo1 (CD95), can activate apoptoticcell death [Chinnaiyan and Dixit, Current Biology, 6:555–562 (1996);Fraser and Evan, Cell; 85:781–784 (1996)]. TNFR1 is also known tomediate activation of the transcription factor, NF-κB [Tartaglia et al.,Cell, 74:845–853 (1993); Hsu et al., Cell, 84:299–308 (1996)]. Inaddition to some ECD homology, these two receptors share homology intheir intracellular domain (ICD) in an oligomerization interface knownas the death domain [Tartaglia et al., supra; Nagata, Cell, 88:355(1997)]. Death domains are also found in several metazoan proteins thatregulate apoptosis, namely, the Drosophila protein, Reaper, and themammalian proteins referred to as FADD/MORT1, TRADD, and RIP [Cleavelandand Ihle, Cell, 81:479–482 (1995)].

Upon ligand binding and receptor clustering, TNFR1 and CD95 are believedto recruit FADD into a death-inducing signalling complex. CD95purportedly binds FADD directly, while TNFR1 binds FADD indirectly viaTRADD [Chinnaiyan et al., Cell, 81:505–512 (1995); Boldin et al., J.Biol. Chem., 270:387–391 (1995); Hsu et al., supra; Chinnaiyan et al.,J. Biol. Chem., 271:4961–4965 (1996)]. It has been reported that FADDserves as an adaptor protein which recruits the Ced-3-related protease,MACHα/FLICE (caspase 8), into the death signalling complex [Boldin etal., Cell, 85:803–815 (1996); Muzio et al., Cell, 85:817–827 (1996)].MACHα/FLICE appears to be the trigger that sets off a cascade ofapoptotic proteases, including the interleukin-1β converting enzyme(ICE) and CPP32/Yama, which may execute some critical aspects of thecell death programme [Fraser and Evan, supra].

It was recently disclosed that programmed cell death involves theactivity of members of a family of cysteine proteases related to the C.elegans cell death gene, ced-3, and to the mammalian IL-1-convertingenzyme, ICE. The activity of the ICE and CPP32/Yama proteases can beinhibited by the product of the cowpox virus gene, cmrA [Ray et al.,Cell, 69:597–604 (1992); Tewari et al., Cell, 81:801–809 (1995)]. Recentstudies show that CrmA can inhibit TNFR1- and CD95-induced cell death[Enari et al., Nature, 375:78–81 (1995); Tewari et al., J. Biol. Chem.,270:3255–3260 (1995)].

As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40 modulatethe expression of proinflammatory and costimulatory cytokines, cytokinereceptors, and cell adhesion molecules through activation of thetranscription factor, NF-κB [Tewari et al., Curr. Op. Genet. Develop.,6:39–44 (1996)]. NF-κB is the prototype of a family of dimerictranscription factors whose subunits contain conserved Rel regions[Verma et al., Genes Develop., 2:2723–2735 (1996); Baldwin, Ann. Rev.Immunol., 14:649–681 (1996)]. In its latent form, NF-κB is complexedwith members of the IκB inhibitor family; upon inactivation of the IκBin response to certain stimuli, released NF-κB translocates to thenucleus where it binds to specific DNA sequences and activates genetranscription.

10. PRO382

Proteases are enzymatic proteins which are involved in a large number ofvery important biological processes in mammalian and non-mammalianorganisms. Numerous different protease enzymes from a variety ofdifferent mammalian and non-mammalian organisms have been bothidentified and characterized, including the serine proteases whichexhibit specific activity toward various serine-containing proteins. Themammalian protease enzymes play important roles in biological processessuch-as, for example, protein digestion, activation, inactivation, ormodulation of peptide hormone activity, and alteration of the physicalproperties of proteins and enzymes.

In light of the important physiological roles played by proteaseenzymes, efforts are currently being undertaken by both industry andacademia to identify new, native protease homologs. Many of theseefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel membrane-boundreceptor proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No. 5,536,637)]. We hereindescribe the identification of novel polypeptides having homology toserine protease enzymes, designated herein as PRO382 polypeptides.

11. PRO545

The ADAM (A Disintegrin And Metalloprotease) family of proteins of whichmeltrin is a member may have an important role in cell interactions andin modulating cellular responses. [see, for example, Gilpin et al., J.Biol. Chem., 273(1):157–166(1998)]. The ADAM protein shave beenimplicated in carcinogenesis. Meltrin-α (ADAM12) is a myoblast geneproduct reported to be required for cell fusion. [Harris et al., J.Cell. Biochem., 67(1):136–142 (1997), Yagami-Hiromasa et al., Nature,377:652–656 (1995)]. The meltrins contain disintegrin andmetalloprotease domains and are implicated in cell adhesive eventsinvolved in development, through the integrin-binding disintegrindomain, but also have an anti-adhesive function through a zinc-dependentmetalloprotease domain. [Alfandari et al., Devel. Biol., 182(2):314–330(1997)]. Given the medical importanc of cell fusion and modulation ofcellular responses in carcinogenesis and other disease mechanisms,efforts are currently being under taken to identify new, native proteinswhich are involved in cell fusion and modulation of cellular responses.We describe herein the identification of a novel polypeptide which hashomology to meltrin, designated herein as PRO545.

12. PRO617

CD24 is a protein that is associated with the cell surface of a varietyof different cells of the mammalian immune system, including forexample, neutrophils, monocytes and some lymphocytes, for example, Blymphocytes. CD24 has been shown to be a ligand for theplatelet-associated surface glycoprotein P-selectin (also known asgranule membrane protein-140 or GMP-140), a glycoprotein that isconstitutively synthesized in both platelets and endothelial cells andbecomes exposed on the surface of platelets when those cells becomeactivated. In this way, P-selectin mediates the calcium-dependentadhesion of activated platelets and endothelial cells to the variouscells of the immune system that express one or more ligands for theP-selectin molecule, particularly CD24. This mechanism allows forrecruitment of immune system cells to locations where they are mostneeded, for example, sites of injury. Thus, there is substantialinterest in identifying novel polypeptides that exhibit homology to thecell surface antigens of the immune system cells. We herein describe theidentification and characterization of a novel polypeptide havinghomology to the CD24 protein, wherein that novel polypeptide is hereindesignated PRO617.

13. PRO700

Protein-disulfide isomerase (PDI) is a catalyst of disulfide formationand isomerization during protein folding. It has two catalytic siteshoused in two domains homologous to thioredoxin, one near the N terminusand the other near the C terminus. [See for example, Gilbert H F,J.Biol.Chem., 47:29399–29402 (1997), Mayfield K J, Science,278:1954–1957 (1997) and Puig et al., J. Biol. Chem., 52:32988–32994(1997)]. PDI is useful for formation of natural type disulfide bonds ina protein which is produced in aprokaryotic cell. (See also, U.S. Pat.Nos. 5,700,659 and 5,700,678).

Thus, PDI and molecules related thereto are of interest, particularlyfor ability to catalyze the formation of disulfide bonds. Moreover,these molecules are generally of interest in the study of redoxreactions and related processes. PDI and related molecules are furtherdescribed in Darby, et al., Biochemistry 34, 11725–11735(1995). Weherein describe the identification and characterization of novelpolypeptides having homology to protein disulfide isomerase, designatedherein as PRO700 polypeptides.

14. PRO702

Conglutinin is a bovine serum protein that was originally described as avertebrate lectin protein and which belongs to the family of C-typelectins that have four characteristic domains, (1) an N-terminalcysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and(4) a carbohydrate recognition domain (CRD). Recent reports havedemonstrated that bovine conglutinin can inhibit hemagglutination byinfluenza A viruses as a result of their lectin properties (Eda et al.,Biochem. J. 316:43–48 (1996)). It has also been suggested that lectinssuch as conglutinin can function as immunoglobulin-independent defensemolecules due to complement-mediated mechanisms. Thus, conglutinin hasbeen shown to be useful for purifying immune complexes in vitro and forremoving circulating immune complexes from patients plasma in vivo (Limet al., Biochem. Biophys. Res. Commun. 218:260–266 (1996)). We hereindescribe the identification and characterization of a novel polypeptidehaving homology to the conglutinin protein, designated herein as PRO702.

15. PRO703

Very-long-chain acyl-CoA synthetase (“VLCAS”) is a long-chain fatty acidtransport protein which is active in the cellular transport of long andvery long chain fatty acids. [see for example, Uchida et al., J Biochem(Tokyo) 119(3):565–571 (1996) and Uchiyama et al., J Biol Chem271(48):30360–30365 (1996). Given the biological importance of fattyacid transport mechanisms, efforts are currently being under taken toidentify new, native proteins which are involved in fatty acidtransport. We describe herein the identification of a novel polypeptidewhich has homology to VLCAS, designated herein as PRO703.

16. PRO705

The glypicans are a family of glycosylphosphatidylinositol(GPI)-anchored proteoglycans that, by virtue of their cell surfacelocalization and possession of heparin sulfate chains, may regulate theresponses of cells to numerous heparin-binding growth factors, celladhesion molecules and extracellular matrix components. Mutations in oneglypican protein cause of syndrome of human birth defects, suggestingthat the glypicans may play an important role in development (Litwack etal., Dev. Dyn. 211:72–87 (1998)). Also, since the glypicans may interactwith the various extracellular matrices, they may also play importantroles in wound healing (McGrath et al., Pathol. 183:251–252 (1997)).Furthermore, since glypicans are expressed in neurons and glioma cells,they may also play an important role in the regulation of cell divisionand survival of cells of the nervous system (Liang et al., J. Cell.Biol. 139:851–864 (1997)). It is evident, therefore, that the glypicansare an extremely important family of proteoglycans. There is, therefore,substantial interest in identifying novel polypeptides having homologyto members of the glypican family. We herein describe the identificationand characterization of a novel polypeptide having homology toK-glypican, designated herein as PRO705.

17. PRO708

Aryl sulfatases are enzymes that exist in a number of differentisoforms, including aryl sulfatase A (ASA), aryl sulfatase B (ASB) andaryl sulfatase C (ASC), and that function to hydrolyze a variety ofdifferent aromatic sulfates. Aryl sulfatases have been isolated from avariety of different animal tissues and microbial sources and theirstructures and functions have been extensively studied (see, e.g.,Nichol and Roy, J. Biochem. 55:643–651 (1964)). ASA deficiency has beenreported to be associated with metachromatic leukodystrophy (MLD) (Gileset al., Prenat. Diagn. 7(4):245–252 (1987) and Herska et al., Am. J.Med. Genet. 26(3):629–635 (1987)). Additionally, other groups havereported that aryl sulfatases have been found in high levels in naturalkiller cells of the immune system and have hypothesized a possible rolefor these enzymes in NK cell-mediated cellular lysis (see, e.g.,Zucker-Franklin et al., Proc. Natl. Acad. Sci. USA 80(22):6977–6981(1983)). Given the obvious physiological importance of the arylsulfatase enzymes, there is a substantial interest in identifying novelaryl sulfatase homolog polypeptides. We herein describe theidentification and characterization of novel polypeptides havinghomology to the aryl sulfatases, wherein these novel polypeptides areherein designated PRO708 polypeptides.

18. PRO320

Fibulin-1 is a cysteine-rich, calcium-binding extracellular matrix (ECM)component of basement membranes and connective tissue elastic fibers andplasma protein, which has four isoforms, A-D, derived from alternativesplicing. Fibulin-1 is a modular glycoprotein with amino-terminalanaphlatoxin-like modules followed by nine epidermal growth factor(EGF)-like modules and, depending on alternative splicing, four possiblecarboxyl termini. Fibulin-2 is a novel extracellular matrix proteinfrequently found in close association with microfibrils containingeither fibronectin or fibrillin. There are multiple forms of fibulin-1that differ in their C-terminal regions that are produced through theprocess of alternative splicing of their precursor RNA. [see for exampleTran et al., Matrix Biol 15(7):479–493 (1997).]

Northern and Western blotting analysis of 16 cell lines established fromtumors formed in athymic mice and malignant cell lines derived frompatients indicate that low expression of fibulin-1D plays a role intumor formation and invasion. [Qing et al., Oncogene, 18:2159–2168(1997)]. Ovarian-cancer cells are characterized by their ability toinvade freely the peritoneal cavity. It has been demonstrated thatestradiol stimulates the proliferation of estrogen-receptor(ER)-positive ovarian-cancer cells, as well as expression of fibulin-1.Studies on the effect of fibulin-1 on motility of the MDA-MB231breast-cancer cell line, indicated inhibition of haptotactic migrationof MDA-MB231 cells, and the authors concluded that fibulin-1 can inhibitcancer cell motility in vitro and therefore has the potential to inhibittumor invasion. [Hayashido et al., Int J Cancer, 75(4):654–658 (1998)]

Thus, fibulin, and molecules related thereto are of interest,particularly for the use of preventing cancer. Moreover, these moleculesare generally of interest in the study of connective tissue andattachment molecules and related mechanisms. Fibulin and relatedmolecules are further described in Adams, et al., J. Mol. Biol.,272(2):226–36 (1997); Kielty and Shuttleworth, Microse. Res. Tech.,38(4):413–27 (1997); and Child. J. Card. Surg. 12(2Supp.):131–5 (1997).

We herein describe the identification and characterization of novelpolypeptides having homology to fibulin, designated herein as PRO320polypeptides.

19. PRO324

Oxidoreductases are enzymes that catalyze a reaction in which twomolecules of a compound interact so that one molecule is oxidized andthe other is reduced, with a molecule of water entering the reaction.There are many different types of oxidoreductase enzymes that play veryimportant physiological roles in the mammalian organism. Some of themost important oxidoreductases include, for example, lyases, lactases,cholesterol oxidases, and the like. These enzymes play roles in suchessential processes as digestion, signal transduction, maintenance ofionic homeostasis, and the like. As such, given that oxidoreductaseenzymes find various essential uses in the mammalian organism, there isa substantial interest in identifying novel oxidoreductase enzymehomologs. We herein describe the identification and characterization ofa novel polypeptide having homology to oxidoreductases, designatedherein as PRO324.

20. PRO351

Prostasin is a novel human serine proteinase purified from human seminalfluid. Immunohistochemical localization reveals that prostasin ispresent in epithelial cells and ducts of the prostate gland. The cDNAfor prostasin has been cloned and characterized. Southern blot analysis,following a reverse transcription polymerase chain reaction, indicatesthat prostasin mRNA is expressed in prostate, liver, salivary gland,kidney, lung, pancreas, colon, bronchus, renal proximal tubular cells,and prostate carcinoma LNCαP cells. Cellular localization of prostasinmRNA was identified within epithelial cells of the human prostate glandby in situ hybridization histochemistry. [See for example, Yu et al., JBiol Chem. (1994) 269(29):18843–18848, and Yu et al., J Biol Chem.(1994) 270(22):13483–13489].

Thus, prostasin, and molecules related thereto are of interest,particularly for the study, diagnosis and treatment of medicalconditions involving the prostate. Prostasin and related molecules arefurther described in Yu et al., Genomics (1996) 32(3):334–340. We hereindescribe the identification and characterization of novel polypeptideshaving homology to prostasin, designated herein as PRO351 polypeptides.

21. PRO352

Butyrophilin is a milk glycoprotein that constitutes more than 40% ofthe total protein associated with the fat globule membrane in mammalianmilk. Expression of butyrophilin mRNA has been shown to correlate withthe onset of milk fat production toward the end pregnancy and ismaintained throughout lactation. Butyrophilin has been identified inbovine, murine and human (see Taylor et al., Biochim. Biophys. Acta1306:1–4 (1996), Ishii et al., Biochim. Biophys. Acta 1245:285–292(1995), Mather et al., J. Dairy Sci. 76:3832–3850 (1993) and Banghart etal., J. Biol. Chem. 273:4171–4179 (1998)) and is a type I transmembraneprotein that is incorporated into the fat globulin membrane. It has beensuggested that butyrophilin may play a role as the principle scaffoldfor the assembly of a complex with xanthine dehydrogenase/oxidase andother proteins that function in the budding and release of milk-fatglobules from the apical surface during lactation (Banghart et al.,supra).

Given that butyrophilin plays an obviously important role in mammalianmilk production, there is substantial interest in identifying novelbutyrophilin homologs. We herein describe the identification andcharacterization of a novel polypeptide having homology to butyrophilin,designated herein as PRO352.

22. PRO381

The immunophilins are a family of proteins that function as receptorsfor immunosuppressant drugs, such as cyclosporin A, FK506, andrapamycin. The immunophilins occur in two separate classes, (1) theFK506binding proteins (FKBPs), which bind to FK506 and rapamycin, and(2) the cyclophilins, which bind to cyclosporin A. With regard to theFK506-binding proteins, it has been reported that the FK506/FKBP complexfunctions to inhibit the activity of the serine/threonine proteinphosphatase 2B (calcineurin), thereby providing immunosuppressantactivity (Gold, Mol. Neurobiol. 15:285–306 (1997)). It has also beenreported that the FKBP immunophilins are found in the mammalian nervoussystem and may be involved in axonal regeneration in the central nervoussystem through a mechanism that is independent of the process by whichimmunosuppression is achieved (Gold, supra). Thus, there is substantialinterest in identifying novel polypeptides having homology to the FKBPimmunophilins. We herein describe the identification andcharacterization of a novel polypeptide having homology to an FKBPimmunophilin protein, designated herein as PRO381.

23. PRO386

Mammalian cell membranes perform very important functions relating tothe structural integrity and activity of various cells and tissues. Ofparticular interest in membrane physiology is the study of transmembraneion channels which act to directly control a variety of physiological,pharmacological and cellular processes. Numerous ion channels have beenidentified including calcium (Ca), sodium (Na) and potassium (K)channels, each of which have been analyzed in detail to determine theirroles in physiological processes in vertebrate and insect cells.

One type of cell membrane-associated ion channel, the sodium channel,plays an extremely important role in a cell's ability to maintain ionichomeostasis as well as transmit intracellular and extracellular signals.Voltage-gated sodium channels in brain neurons have been shown to becomplexes of a pore-forming alpha unit with smaller beta-1 and beta-2subunits (Isom et al., Cell 83:433–442 (1995)). Given the obviousimportance of sodium channels in cellular homeostasis and otherimportant physiological functions, there is a significant interest inidentifying novel polypeptides having homology to sodium channelsubunits. We herein describe the identification and characterization ofa novel polypeptide having homology to the beta-2 subunit of the ratsodium channel, designated herein as PRO386.

24. PRO540

Lecithin-cholesterol acyltransferase (“LCAT”), also known asphosphatidylcholine-sterol acyltransferase is a key enzyme in theintravascular metabolism of high density lipoproteins, specifically inthe process of cholesterol metabolism. [see, for example, Brousseau etal., J. Lipid Res., 38(12):2537–2547 (1997), Hill et al., Biochem. J.,294:879–884 (1993), and Drayna et al., Nature 327 (6123):632–634(1987)]. Given the medical importance of lipid metabolism, efforts arecurrently being under taken to identify new, native proteins which areinvolved in lipid transport. We describe herein the identification of anovel polypeptide which has homology to LCAT, designated herein asPRO540.

25. PRO615

Synaptogyrin is a synaptic vesicle protein that is uniformly distributedin the nervous system. The cDNA encoding synaptogyrin has been clonedand sequenced and the sequence predicts a protein with a molecular massof 25,900 D with four membrane-spanning domains. Synaptogyrin has beenimplicated in membrane traffic to and from the plasma membrane. Steniuset al., J. Cell. Biol. 131(6–2):1801–1809 (1995). In addition, a novelisoform of synaptogyrin called cellugyrin exhibits sequence identitywith synaptogyrin. In rat tissues, cellugyrin and synaptogyrins areexpressed in mirror image patterns. Cellugyrin is ubiquitously presentin all tissues tested with the lowest levels in brain tissue, whereassynaptogyrin protein is only detectable in brain. In rat tissues,cellugyrin and synaptogyrins are expressed in mirror image patterns. Thesynaptic vesicle protein synaptogyrin may be a specialized version of aubiquitous protein, cellugyrin, with the two proteins sharing structuralsimilarity but differing in localization. This finding supports theemerging concept of synaptic vesicles as the simplified and specializedform of a generic trafficking organelle. [Janz et al., J. Biol. Chem.273(5):2851–2857 (1998)]. The sequence for cellugyrin derived from theNorway rat, Rattus norvegicus has been deposited in the Genbank databaseon 23 Dec. 1997, designated accession number AF039085. See also, Janz etal., J. Biol. Chem. 273 (1998), in press.

Given the medical importance of synaptic transmission, efforts arecurrently being under taken to identify new, native proteins that may bepart of a simplified and specialized generic trafficking organelle inthe form of synaptic vesicles. We describe herein the identification ofa novel polypeptide which has homology to synaptogyrin, designatedherein as PRO615.

26. PRO618

Enteropeptidase is a key enzyme in the intestinal digestion cascadespecifically cleaves the acidic propeptide from trypsinogen to yieldactive trypsin. This cleavage initiates a cascade of proteolyticreactions leading to the activation of many pancreatic zymogens.

See, for example, Matsushima et al., J. Biol. Chem. 269(31):19976–19982(1994), Kitamoto et al., Proc. Nat. Acad. Sci., 91(16):7588–7592 (1994).Enterokinase (enteropeptidase) is a related to mammalian serineproteases involved in digestion, coagulation, and fibrinolysis. LaVallieet al., J Biol Chem., 268(31):23311–23317 (1993).

Given the medical importance of digestive processes, efforts arecurrently being under taken to identify new, native proteins that may beinvolved in digestion, coagulation, and fibrinolysis. We describe hereinthe identification of a novel polypeptide which has homology toenteropeptidase, designated herein as PRO618.

27. PRO719

Lipoprotein lipase is a key enzyme that mediates the hydrolysis oftriglycerides and phospholipids present in circulating plasmalipoproteins (Dugi et al., J. Biol. Chem. 270:25396–25401 (1995)).Moreover, lipoprotein lipase has been shown to mediate the uptake oflipoproteins into cells, wherein cellular uptake of lipoproteins isinitiated by binding of lipoprotein lipase to cell surface proteoglycansand to the low density lipoprotein (LDL) receptor-related protein (Krappet al., J. Lipid Res. 36:2362–2373 (1995)). Thus, it is clear thatlipoprotein lipase plays an extremely important role in lipoprotein andcholesterol metabolism. There is, therefore, substantial interest inidentifying novel polypeptides that share sequence homology and/orbiological activity with lipoprotein lipase. We herein describe theidentification and characterization of a novel polypeptide havingsequence homology to lipoprotein lipase H, designated herein as PRO719.

28. PRO724

The low density lipoprotein (LDL) receptor is a membrane-bound proteinthat plays a key role in cholesterol homeostasis, mediating cellularuptake of lipoprotein particles by high affinity binding to its ligands,apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of theLDL receptor contains 7 cysteine-rich repeats of approximately 40 aminoacids, wherein each repeat contains 6 cysteines, which form 3intra-repeat disulfide bonds. These unique structural features providethe LDL receptor with its ability to specifically interact with apoB-100 and apoE, thereby allowing for transport of these lipoproteinparticles across cellular membranes and metabolism of their components.Soluble fragments containing the extracellular domain of the LDLreceptor have been shown to retain the ability to interact with itsspecific lipoprotein ligands (Simmons et al., J. Biol. Chem.272:25531–25536 (1997)). Thus, it is clear that the LDL receptor isintimately involved in important physiological activities related tocholesterol metabolism. As such, there is substantial interest inidentifying novel LDL receptor homolog proteins. We herein describe theidentification and characterization of a novel polypeptide havinghomology to the human LDL receptor protein, designated herein as PRO724.

29. PRO772

Expression of the human gene A4 is enriched in the colonic epitheliumand is transcriptionally activated on differentiation of colonicepithelial cells in vitro (Oliva et al., Arch. Biochem. Biophys.302:183–192 (1993) and Oliva et al., Am. J. Physiol. 272: C957–C965(1997)). A4 cDNA contains an open reading frame that predicts apolypeptide of approximately 17 kilodaltons in size. Hydropathy analysisof the A4 protein revealed four putative membrane-spanningalpha-helices. Immunocytochemical studies of cells expressing A4 proteinindicated that expression is localized to the endoplasmic reticulum. Thefour membrane-spanning domains and the biophysical characteristics ofthe A4 protein suggest that it belongs to a family of integral membraneproteins called proteolipids, some of which multimerize to form ionchannels. In fact, preliminary evidence has suggested that A4 may itselfmultimerize and take on the properties of an ion channel (Oliva et al.,Am. J. Physiol. 272:C957–C965 (1997)). Given the importance of ionchannels in maintaining cellular homeostasis, there is a significantinterest in identifying novel polypeptides having homology to known andputative ion channels We herein describe the identification andcharacterization of a novel polypeptide having homology to the putativeion channel protein, A4, designated herein as PRO772.

30. PRO852

Proteases are enzymatic proteins which are involved in a large number ofvery important biological processes in mammalian and non-mammalianorganisms. Numerous different protease enzymes from a variety ofdifferent mammalian and non-mammalian organisms have been bothidentified and characterized. The mammalian protease enzymes playimportant roles in many different biological processes including, forexample, protein digestion, activation, inactivation, or modulation ofpeptide hormone activity, and alteration of the physical properties ofproteins and enzymes.

In light of the important physiological roles played by proteaseenzymes, efforts are currently being undertaken by both industry andacademia to identify new, native protease homologs. Many of theseefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel secreted andmembrane-bound receptor proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No.5,536,637)]. We herein describe the identification of novel polypeptideshaving homology to various protease enzymes, designated herein as PRO852polypeptides.

31. PRO853

Studies have reported that the redox state of the cell is an importantdeterminant of the fate of the cell. Furthermore, reactive oxygenspecies have been reported to be cytotoxic, causing inflammatorydisease, including tissue necrosis, organ failure, atherosclerosis,infertility, birth defects, premature aging, mutations and malignancy.Thus, the control of oxidation and reduction is important for a numberof reasons, including the control and prevention of strokes, heartattacks, oxidative stress, hypertension and may be associated with thedevelopment of malignancies. The levels of antioxidant enzymes, such asreductases, which catalyze the conversion of reactive oxygen species towater have been shown to be low in cancer cells. In particular,malignant prostate epithelium may have lowered expression of suchantioxidant enzymes [Baker et., Prostate 32(4):229–233 (1997)]. In thisregard, reductases, are of interest. In addition, the transcriptionfactors, NF-kappa B and AP-1, are known to be regulated by redox stateand to affect the expression of a large variety of genes thought to beinvolved in the pathogenesis of AIDS, cancer, atherosclerosis anddiabetic complications. Publications further describing this subjectmatter include Engman et al., Anticancer Res. (Greece), 17:4599–4605(1997), Kelsey, et al., Br. J. Cancer, 76(7):852–4 (1997); Friedrich andWeiss, J. Theor. Biol., 187(4):529–40 (1997) and Pieulle, et al., J.Bacteriol., 179(18):5684–92 (1997). Given the physiological importanceof redox reactions in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in redox reactions. Wedescribe herein the identification of a novel prostate specificpolypeptide which has sequence similarity to reductase, designatedherein as PRO853.

32. PRO860

Neurofascin is a member of the L1 subgroup of the cellular adhesionmolecule (“CAM”) family of nervous system adhesion molecules and isinvolved in cellular aggregation. Cell-cell recognition and patterningof cell contacts have a critical role in mediating reversible assemblyof a wide variety or transcellular complexes in the nervous system. Cellinteractions may be regulated through modulation of ankyrin binding toneurofascin. See, for example, Tuvia et al., Proc. Nat Acad. Sci.,94(24) 12957–12962 (1997). Neurofascin has been described as a member ofthe L1 subgroup of the immunoglobulin superfamily implicated in neuriteextension during embryonic development for which numerous isoforms havebeen detected at various stages of development. See also Hassel et al.,J. Biol. Chem., 272(45) 28742–28749 (1997), Grumet., Cell. Tissue Res.290(2) 423–428 (1997), Garver et al., J. Cell. Biol., 137:703–714(1997), and Lambert et al., J. Neurosci., 17:7025–7–36 (1997).

Given the physiological importance of cellular adhesion molecules anddevelopment of the nervous system in vivo, efforts are currently beingunder taken to identify new, native proteins which are involved inregulation of cellular interactions in the nervous system. We describeherein the identification and characterization of a novel polypeptidewhich has sequence similarity to neurofascin, designated herein asPRO860.

33. PRO846

The CMRF35 monoclonal antibody was used to identify a cell membraneantigen, designated CMRF35, which is present on the surface ofmonocytes, neutrophils, a proportion of peripheral blood T and Blymphocytes and lymphocytic cell lines. The CMRF35 cDNA encodes a novelintegral membrane glycoprotein member of the immunoglobulin (Ig) genesuperfamily. The molecule comprises (a) a single extracellular Igvariable domain remarkably similar to the Fc receptor for polymeric IgAand IgM, (b) a membrane-proximal domain containing a high proportion ofproline, serine and threonine residues that was predicted to be heavilyO-glycosylated, (c) an unusual transmembrane anchor that contained aglutamic acid and a proline residue and (d) a short cytoplasmic tail.Transcripts encoding the CMRF35 protein have been detected in earlymonocytic cell lines, in peripheral blood T cells and in some Blymphoblastoid cell lines, confirming the results of immunocytologicalstaining. Jackson et al., Eur. J. Immunol. 22(5):1157–1163 (1992).CMRF-35 molecules are differentially expressed in hematopoietic cells,and the expression of the antigen was shown to be markedly influenced bystimulation with mitogens and cytokines. See, for example, Clark et al.,Exp. Hematol. 25(8):759 (1997), Daish et al., Immunol. 79(1):55–63(1993), and Clark et al., Tissue Antigens 48:461 (1996).

Given the physiological importance of the immune system and antigensassociated with various immune system cells, efforts are currently beingunder taken to identify new, native proteins which are expressed onvarious cells of the immune system. We describe herein theidentification of a novel polypeptide which has sequence similarity toCMRF35, designated herein as PRO846.

34. PRO862

Lysozyme is a protein which is widely distributed in several humantissues and secretions including milk, tears and saliva. It has beendemonstrated to hydrolyze linkages between N-acetylglucosamines. It hasbeen demonstrated to be an inhibitor of chemotaxis and of the productionof toxic oxygen free radicals and may also have some role in thecalcification process. As such, there is substantial interest inidentifying novel polypeptides having homology to lysozyme. We describeherein the identification of a novel polypeptide which has sequencesimilarity to lysozyme.

35. PRO864

Wnt-4 is a secreted glycoprotein which correlates with, and is requiredfor, kidney tubulogenesis. Mice lacking Wnt-4 activity fail to formpretubular cell aggregates; however, other aspects of mesenchymal andureteric development are unaffected. Thus, Wnt-4 appears to act as anautoinducer of the mesenchyme to epithelial transition that underliesnephron development. Stark et al., Nature; 372(6507):679–683 (1994). Inaddition, members of the Wnt gene family code for cysteine-rich,secreted proteins, which are differentially expressed in the developingbrain and possibly act as intercellular signaling molecules. A Wnt gene,e.g., Wnt-1 is known to be essential for specification of the midbraincell fate. Yoshioka et al., Biochem. Biophys. Res. Commun.203(3):1581–1588 (1994). Several member of the Wnt family of secretedfactors are strongly implicated as regulators of mammary cellular growthand differentiation. Shimizu et al., Cell Growth Differ. 8(12)1349–1358. Wnt-4 is normally expressed in early pregnancy. Wnt-4 maytherefore be a local signal driving epithelial branching in pregnancy.Edwards P A, Biochem Soc Symp. 63:21–34 (1998). See also, Lipschutz J H,Am. J. Kidney Dis. 31(3):383–397, (1998). We describe herein theidentification and characterizaton of a novel polypeptide which hassequence similarity to Wnt-4, designated herein as PRO864.

36. PRO792

At least two cell-derived signals have been shown to be necessary forthe induction of immunoglobulin isotype switching in Bells. The firstsignal is given by either of the soluble lymphokines, interleukin (IL)-4or IL-13, which induce germline epsilon transcript expression, but thisalone is insufficient to trigger secretion of immunoglobulin E (IgE).The second signal is provided by a physical interaction between B-cellsand activated Tells, basophils and mast cells, and it has been shownthat the CD40ICD40 ligand pairing is crucial for mediating IgEsynthesis. Additionally, amongst the numerous pairs of surface adhesionmolecules that are involved in IgE synthesis, the CD23/CD21 pair appearsto play a key role in the generation of IgE. CD23 is a protein that ispositively and negatively regulated by factors which increase ordecrease IgE production, respectively. Antibodies to CD23 have beenshown to inhibit IL-4-induced human IgE production in vitro and toinhibit antigen-specific IgE responses in a rat model, in an isotypeselective manner (Bonnefoy et al., Eur. Respir. J. Suppl. 22:63S–66S(1996)). CD23 interacts with CD21 on B-cells, preferentially driving IgEproduction. Given that the CD23 protein plays an extremely importantrole in the induction of a mammalian IgE response, there is significantinterest in identifying novel polypeptides having homology to CD23. Weherein describe the identification and characterization of a novelpolypeptide having homology to CD23, designated herein as PRO792.

37. PRO866

Mindin and spondin proteins are secreted proteins that are structurallyrelated to one another and which have been identified in a variety oforganisms. For example, Higashijima et al., Dev Biol. 192:211–227 (1997)have reported the identification of spondin and mindin expression infloor plate cells in the zebrafish embryonic axis, thereby suggestingthat mindin and spondin proteins play important roles in embryonicdevelopment. This same group has reported that mindin and spondinproteins function as extracellular matrix proteins that have a highaffinity for the basal lamina. (Id.). It has been reported thatF-spondin is a secreted protein that promotes neural adhesion andneurite extension (Klar et al., Cell 69:95–110 (1992) and that M-spondinis an extracellular matrix protein that localizes to muscle attachmentsites in Drosophila (Umemiya et al., Dev. Biol. 186:165–176 (1997)).Thus, there is significant interest in identifying novel polypeptideshaving homology to the mindin and spondin proteins. We herein describethe identification and characterization of a novel polypeptide havinghomology to mindin2 and mindin1, designated herein as PRO866.

38. PRO871

Cyclophilins are a family of proteins that bind to cyclosporin A andpossess peptidyl-prolyl cis-trans isomerase activity (Sherry et al.,Proc. Natl. Acad. Sci. USA 95:1758–1763 (1998)). In addition,cyclophilins are secreted by activated cells and act in a cytokine-likemanner, presumably via signaling through a cell surface cyclophilinreceptor. Host cell-derived cyclophilin A has been shown to beincorporated into HIV-1 virions and its incorporation has been shown tobe essential for viral infectivity. Thus, one or more the cyclophilinsmay be directly associated with HIV-1 infectivity. Given the obviousimportance of the cyclophilin proteins, there is substantial interest inidentifying novel polypeptides which have sequence homology to one ormore of the cyclophilin proteins. We herein describe the identificationand characterization of a novel polypeptide having homology tocyclophilin-like protein CyP-60, designated herein as PRO871.

39. PRO873

Enzymatic proteins play important roles in the chemical reactionsinvolved in the digestion of foods, the biosynthesis of macromolecules,the controlled release and utilization of chemical energy, and otherprocesses necessary to sustain life. Enzymes have also been shown toplay important roles in combating various diseases and disorders. Forexample, liver carboxylesterases have been reported to assist insensitizing human tumor cells to the cancer prodrugs. Danks et al.,report that stable expression of the cDNA encoding a carboxylesterase inRh30 human rhabdomyosarcoma cells increased the sensitivity of the cellsto the CPT-11 cancer prodrug 8.1-fold. Cancer Res. (1998) 58(1):20–22.The authors propose that this prodrug/enzyme combination could beexploited therapeutically in a manner analogous to approaches currentlyunder investigation with the combinations of ganciclovir/herpes simplexvirus thymidine kinase and 5-fluorocytosine/cytosine deaminase. van Peltet al. demonstrated that a 55 kD human liver carboxylesterase inhibitsthe invasion of Plasmodium falciparum malaria sporozoites into primaryhuman hepatocytes in culture. J Hepatol (1997) 27(4):688–698.

Carboxylesterases have also been found to be of importance in thedetoxification of drugs, pesticides and other xenobiotics. Purifiedhuman liver carboxylesterases have been shown to be involved in themetabolism of various drugs including cocaine and heroin. Prindel et al.describe the purification and cloning of a broad substrate specificityhuman liver carboxylesterase which catalyzes the hydrolysis of cocaineand heroin and which may play an important role in the degradation ofthese drugs in human tissues. J. Biol. Chem. (1997)6:272(23):14769–14775. Brzenzinski et al. describe a spectrophotometriccompetitive inhibition assay used to identify drug or environmentalesters that are metabolized by carboxylesterases. Drug Metab Dispos(1997) 25(9):1089–1096.

In light of the important physiological roles played bycarboxylesterases, efforts are being undertaken by both industry andacademia to identify new, native carboxylesterase homologs. We hereindescribe the identification and characterization of a novel polypeptidehaving homology to carboxylesterase, designated herein as PRO873.

40. PRO940

CD33 is a cell-surface protein that is a member of the sialoadhesinfamily of proteins that are capable of mediating sialic-acid dependentbinding with distinct specificities for both the type of sialic acid andits linkage to subterminal sugars. CD33 is specifically expressed inearly myeloid and some monocyte cell lineages and has been shown to bestrongly associated with various myeloid tumors including, for example,acute non-lymphocytic leukemia (ANLL). As such, CD33 has been suggestedas a potential target for the treatment of cancers associated with highlevel expression of the protein. There is, therefore, significantinterest in the identification of novel polypeptides having homology toCD33. In fact, one CD33 homolog (designated CD33L) has already beenidentified and described (see Takei et al., Cytogenet. Cell Genet.78:295–300 (1997)). We herein describe the identification of anothernovel polypeptide having homology to CD33, designated herein as PRO940.The novel polypeptide described herein also exhibits significanthomology to the human OB binding proteins designated HSU71382_(—)1 andHSU71383_(—)1 in the Dayhoff database (version 35.45 SwissProt 35).

41. PRO941

Cadherins are a large family of transmembrane proteins. Cadherinscomprise a family of calcium-dependent glycoproteins that function inmediating cell-cell adhesion in virtually all solid tissues ofmulticellular organisms. At least cadherins 1–13 as well as types B, T,EP, M, N, P and R have been identified and characterized. Among thefunctions cadherins are known for, with some exceptions, are thatcadherins participate in cell aggregation and are associated withcell-cell adhesion sites. Recently, it has been reported that while allcadherins share multiple repeats of a cadherin specific motif believedto correspond to folding of extracellular domains, members of thecadherin superfamily have divergent structures and, possibly, functions.In particular it has been reported that members of the cadherinsuperfamily are involved in signal transduction. See, Suzuki, J. CellBiochem., 61(4):531–542 (1996). Cadherins are further described inTanihara et al., J. Cell Sci., 107(6):1697–1704 (1994), Aberle et al.,J. Cell Biochem., 61(4):514–523 (1996) and Tanihara et al., Cell Adhes.Commun., 2(1):15–26 (1994). We herein describe the identification andcharacterization of a novel polypeptide having homology to a cadherinprotein, designated herein as PRO941.

42. PRO944

Clostridium perfringens enterotoxin (CPE) is considered to be thevirulence factor responsible for causing the symptoms of C. perfringenstype A food poisoning and may also be involved in other human andveterinary illnesses (McClane, Toxicon. 34:1335–1343 (1996)). CPEcarries out its adverse cellular functions by binding to anapproximately 50 kD cell surface receptor protein designated theClostridium perfringens enterotoxin receptor (CPE-R) to form anapproximately 90,000 kD complex on the surface of the cell. cDNAsencoding the CPE-R protein have been identified characterized in bothhuman and mouse (Katahira et al., J. Cell Biol. 136:1239–1247 (1997) andKatahira et al., J. Biol. Chem. 272:26652–26658 (1997)). Since the CPEtoxin has been reported to cause a variety of illnesses in mammalianhosts and those illnesses are initiated by binding of the CPE toxin tothe CPE-R, there is significant interest in identifying novel CPE-Rhomologs. We herein describe the identification and characterization ofa novel polypeptide having homology to the CPE-R, designated herein asPRO944.

43. PRO983

Membrane-bound proteins include not only cell-surface membrane-boundproteins, but also proteins that are found on the surface ofintracellular vesicles. These vesicles are involved in exocytosis, whichis the fusion of secretory vesicles with the cellular plasma membrane,and have two main functions. One is the discharge of the vesiclecontents into the extracellular space, and the second is theincorporation of new proteins and lipids into the plasma membraneitself. Exocytosis can be either constitutive or regulated. Alleukaryotic cells exhibit constitutive exocytosis, which is marked by theimmediate fusion of the secretory vesicle after formation. In contrast,regulated exocytosis results in the accumulation of the secretoryvesicles that fuse with the plasma membrane upon receipt of anappropriate signal by vesicle-associated membrane proteins. Usually,this signal is an increase in the cytosolic free Ca²⁺ concentration.However, regulated exocytosis that is independent of Ca²⁺ has beenreported (see, e.g. Fujita-Yoshigaki et al. J. Biol. Chem. (1996)31:271(22):13130–13134). Regulated exocytosis is chemical to manyspecialized cells, including neurons (neurotransmitter release fromsynaptic vesicles), adrenal chromaffin cells (adrenaline secretion),pancreatic acinar cells (digestive enzyme secretion), pancreatic β-cells(insulin secretion), mast cells (histamine secretion), mammary cells(milk protein secretion), sperm (enzyme secretion), egg cells (creationof fertilization envelope) and adipocytes (insertion of glucosetransporters into the plasma membrane).

Disorders involving exocytosis are known. For example, inflammatorymediator release from mast cells leads to a variety of disorders,including asthma. Similarly, Chediak-Higashi Syndrome (CHS) is a rareautosomal recessive disease in which neutrophils, monocytes andlymphocytes contain giant cytoplasmic granules. Accordingly, theproteins involved in exocytosis are of paramount interest and effortsare being undertaken by both industry and academia to identify new,vesicle-associated proteins. For example, Skehel et al. identified a33-kilodalton membrane protein in Aplysia, termed VAP-33, which isrequired for the exocytosis of neurotransmitter. Science (1995)15:269(5230):1580–1583, and Neuropharmacology (1995) 34(11):1379–1385.Many efforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel vesicle-associatedmembrane proteins. It is an object of the invention to provide proteinshaving homology to the vesicle associated protein, VAP-33, designatedherein as PRO983.

44. PRO1057

Proteases are enzymatic proteins which are involved in a large number ofvery important biological processes in mammalian and non-mammalianorganisms. Numerous different protease enzymes from a variety ofdifferent mammalian and non-mammalian organisms have been bothidentified and characterized. The mammalian protease enzymes playimportant roles in many different biological processes including, forexample, protein digestion, activation, inactivation, or modulation ofpeptide hormone activity, and alteration of the physical properties ofproteins and enzymes.

In light of the important physiological roles played by proteaseenzymes, efforts are currently being undertaken by both industry andacademia to identify new, native protease homologs. Many of theseefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel secreted proteins.Examples of screening methods and techniques are described in theliterature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108–7113 (1996); U.S. Pat. No. 5,536,637)]. We herein describe theidentification of novel polypeptides having homology to various proteaseenzymes, designated herein as PRO1057 polypeptides.

45. PRO1071

Thrombospondin-1 is a trimeric high molecular weight glycoprotein thatis released from platelet alpha-granules in response to thrombinstimulation and that is also a transient component of the extracellularmatrix in developing and repairing tissues (Adams, Int. J. Biochem. CellBiol. 29:861–865 (1997) and Qian et al., Proc. Soc. Exp. Biol. Med.212:199–207 (1996)). A variety of factors regulate thrombospondinexpression and the protein is degraded by both extracellular andintracellular routes. Thrombospondin-I functions as a cell adhesionmolecule and also modulates cell movement, cell proliferation, neuriteoutgrowth and angiogenesis. As such, there is substantial interest inidentifying novel polypeptides having homology to thrombospondin. Weherein describe the identification and characterization of a novelpolypeptide having homology to thrombospondin, designated herein asPRO1071.

46. PRO1072

Studies have reported that the redox state of the cell is an importantdeterminant of the fate of the cell. Furthermore, reactive oxygenspecies have been reported to be cytotoxic, causing inflammatorydisease, including tissue necrosis, organ failure, atherosclerosis,infertility, birth defects, premature aging, mutations and malignancy.Thus, the control of oxidation and reduction is important for a numberof reasons, including the control and prevention of strokes, heartattacks, oxidative stress, hypertension and may be associated with thedevelopment of malignancies. The levels of antioxidant enzymes, such asreductases, which catalyze the conversion of reactive oxygen species towater have been shown to be low in cancer cells. In particular,malignant prostate epithelium may have lowered expression of suchantioxidant enzymes [Baker et al., Prostate 32(4):229–233 (1997)]. Inthis regard, reductases, are of interest. In addition, the transcriptionfactors, NF-kappa B and AP-1, are known to be regulated by redox stateand to affect the expression of a large variety of genes thought to beinvolved in the pathogenesis of AIDS, cancer, atherosclerosis anddiabetic complications. Publications further describing this subjectmatter include Engman et al., Anticancer Res. (Greece), 17:4599–4605(1997), Kelsey, et al., Br. J. Cancer, 76(7):852–854 (1997); Friedrichand Weiss, J. Theor. Biol., 187(4):529–40 (1997) and Pieulle, et al., J.Bacteriol., 179(18):5684–92 (1997). Given the physiological importanceof redox reactions in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in redox reactions. Wedescribe herein the identification of a novel polypeptide which hassequence similarity to reductase enzymes, designated herein as PRO1072.

47. PRO1075

Protein disulfide isomerase is an enzymatic protein which is involved inthe promotion of correct refolding of proteins through the establishmentof correct disulfide bond formation. Protein disulfide isomerase wasinitially identified based upon its ability to catalyze the renaturationof reduced denatured RNAse (Goldberger et al., J. Biol. Chem.239:1406–1410 (1964) and Epstein et al., Cold Spring Harbor Symp. Quant.Biol. 28:439–449 (1963)). Protein disulfide isomerase has been shown tobe a resident enzyme of the endoplasmic reticulum which is retained inthe endoplasmic reticulum via a -KDEL or -HDEL amino acid sequence atits C-terminus.

Given the importance of disulfide bond-forming enzymes and theirpotential uses in a number of different applications, for example inincreasing the yield of correct refolding of recombinantly producedproteins, efforts are currently being undertaken by both industry andacademia to identify new, native proteins having homology to proteindisulfide isomerase. Many of these efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel protein disulfide isomerase homologs. Examples of screeningmethods and techniques are described in the literature [see, forexample, Klein et al., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S.Pat. No. 5,536,637)]. We herein describe a novel polypeptide havinghomology to protein disulfide isomerase, designated herein as PRO1075.

48. PRO181

In Drosophila, the dorsal-ventral polarity of the egg chamber depends onthe localization of the oocyte nucleus and the gurken RNA to thedorsal-anterior corner of the oocyte. Gurken protein presumably acts asa ligand for the drosophila EGF receptor (torpedo/DER) expressed in thesomatic follicle cells surrounding the oocyte. Cornichon is a generequired in the germline for dorsal-ventral signaling (Roth et al., Cell81:967–978 (1995)). Cornichon, gurken and torpedo also function in anearlier signaling event that establishes posterior follicle cell fatesand specifies the anterior-posterior polarity of the egg chamber.Mutations in any or all of these genes prevent the formation of acorrectly polarized microtubule cytoskeleton required for properlocalization of the anterior and posterior determinants bicoid and oskarand for the asymmetric positioning of the oocyte nucleus. Thus, it isclear that the cornichon gene product plays an important role in earlydevelopment. We herein describe the identification and characterizationof a novel polypeptide having homology to the cornichon protein,designated herein as PRO181.

49. PRO195

Efforts are currently being undertaken to identify and characterizenovel transmembrane proteins. We herein describe the identification andcharacterization of a novel transmembrane polypeptide, designated hereinas PRO195.

50. PRO865

Efforts are currently being undertaken to identify and characterizenovel secreted proteins. We herein describe the identification andcharacterization of a novel secreted polypeptide, designated herein asPRO865.

51. PRO827

VLA-2 is an cell-surface integrin protein that has been identified andcharacterized in a number of mammalian organisms, including both mouseand human. VLA-2 has been shown to be a receptor on the surface of cellsfor echovirus-1 (EV-1) which mediates infection of VLA-2-expressingcells by EV-1 (Zhang et al., Virology 235(2):293–301 (1997) andBergelson et al., Science 255:1718–1720 (1992)). VLA-2 has also beenshown to mediate the interaction of collagen with endothelium during invitro vascular tube formation (Jackson et al., Cell Biol. Int.18(9):859–867 (1994)). Various other integrin proteins that sharevarious degrees of amino acid sequence homology with VLA-2 have beenidentified and characterized in a variety of mammalian organism. Theseintegrins have been reported to play important roles in a variety ofdifferent physiological functions. Therefore, there is significantinterest in identifying novel polypeptides having homology to one ormore of the integrin proteins. We herein describe the identification andcharacterization of a novel polypeptide having homology to VLA-2integrin protein, designated herein as PRO827.

52. PRO1114

Many important cytokine proteins have been identified and characterizedand shown to signal through specific cell surface receptor complexes.For example, the class II cytokine receptor family (CRF2) includes theinterferon receptors, the interleukin-10 receptor and the tissue factorCRFB4 (Spencer et al., J. Exp. Med. 187:571–578 (1998) and Kotenko etal., EMBO J. 16:5894–5903 (1997)). Thus, the multitude of biologicalactivities exhibited by the various cytokine proteins is absolutelydependent upon the presence of cytokine receptor proteins on the surfaceof target cells. There is, therefore, a significant interest inidentifying and characterizing novel polypeptides having homology to oneor more of the cytokine receptor family. We herein describe theidentification and characterization of a novel polypeptide havinghomology to cytokine receptor family-4 proteins, designated herein asPRO1117.

Interferons (IFNs) encompass a large family of secreted proteinsoccurring in vertebrates. Although they were originally named for theirantiviral activity, growing evidence supports a critical role for IFNsin cell growth and differentiation (Jaramillo et al., CancerInvestigation 13(3):327–338 (1995)). IFNs belong to a class of negativegrowth factors having the ability to inhibit the growth of a widevariety of cells with both normal and transformed phenotypes. IFNtherapy has been shown to be beneficial in the treatment of humanmalignancies such as Karposi's sarcoma, chronic myelogenous leukemia,non-Hodgkin's lymphoma, and hairy cell leukemia as well as in thetreatment of infectious diseases such as hepatitis B (Gamliel et al.,Scanning Microscopy 2(1):485–492 (1988), Einhorn et al., Med. Oncol. &Tumor Pharmacother. 10:25–29 (1993), Ringenberg et al., MissouriMedicine 85(1):21–26 (1988), Saracco et al., Journal of Gastroenterologyand Hepatology 10:668–673 (1995), Gonzalez-Mateos et al.,Hepato-Gastroenterology 42:893–899 (1995) and Malaguarnera et al.,Pharmacotherapy 17(5):998–1005 (1997)).

Interferons can be classified into two major groups based upon theirprimary sequence. Type I interferons, IFN-α and IFN-β, are encoded by asuperfamily of intronless genes consisting of the IFN-α gene family anda single IFN-β gene that are thought to have arisen from a commonancestral gene. Type I interferons may be produced by most cell types.Type II IFN, or IFN-γ, is restricted to lymphocytes (T cells and naturalkiller cells) and is stimulated by nonspecific T cell activators orspecific antigens in vivo.

Although both type I and type II IFNs produce similar antiviral andantiproliferative effects, they act on distinct cell surface receptors,wherein the binding is generally species specific (Langer et al.,Immunol. Today 9:393–400 (1988)). Both IFN-α and IFN-β bindcompetitively to the same high affinity type I receptor, whereas IFN-γbinds to a distinct type II receptor. The presence and number of IFNreceptors on the surface of a cell does not generally reflect thesensitivity of the cell to IFN, although it is clear that the effects ofthe IFN protein is mediated through binding to a cell surface interferonreceptor. As such, the identification and characterization of novelinterferon receptor proteins is of extreme interest.

We herein describe the identification and characterization of novelinterferon receptor polypeptides, designated herein as “PRO1114interferon receptor” polypeptides. Thus, the PRO1114 polypeptides of thepresent invention represents a novel cell surface interferon receptor.

53. PRO237

Carbonic anhydrase is an enzymatic protein that which aids carbondioxide transport and release in the mammalian blood system bycatalyzing the synthesis (and the dehydration) of carbonic acid from(and to) carbon dioxide and water. Thus, the actions of carbonicanhydrase are essential for a variety of important physiologicalreactions in the mammal. As such, there is significant interest in theidentification and characterization of novel polypeptides havinghomology to carbonic anhydrase. We herein describe the identificationand characterization of a novel polypeptide having homology to carbonicanhydrase, designated herein as PRO237.

54. PRO541

Numerous trypsin inhibitory proteins have been identified andcharacterized (see, e.g., Yamakawa et al., Biochim. Biophys. Acta1395:202–208 (1998) and Mizuki et al., Mammalian Genome 3:274–280(1992)). Trypsin inhibitor proteins play important roles in a variety ofdifferent physiological and biological pathways and are specificallyinvolved in such processes as the regulation of protein degradation,digestion, and the like. Given the important roles played by suchenzymatic proteins, there is significant interest in identifying andcharacterizing novel polypeptides having homology to known trypsininhibitor proteins. We herein describe the identification andcharacterization of a novel polypeptide having homology to a trypsininhibitor protein, designated herein as PRO541.

55. PRO273

Leukocytes include monocytes, macrophages, basophils, and eosinophilsand play an important role in the immune response. These cells areimportant in the mechanisms initiated by T and/or B lymphocytes andsecrete a range of cytokines which recruit and activate otherinflammatory cells and contribute to tissue destruction.

Thus, investigation of the regulatory processes by which leukocytes moveto their appropriate destination and interact with other cells iscritical. Currently, leukocytes are thought to move from the blood toinjured or inflamed tissues by rolling along the endothelial cells ofthe blood vessel wall. This movement is mediated by transientinteractions between selectins and their ligands. Next, the leukocytemust move through the vessel wall and into the tissues. This diapedesisand extravasation step involves cell activation which promotes a morestable leukocyte-endothelial cell interaction, again mediated byintegrins and their ligands.

Chemokines are a large family of structurally related polypeptidecytokines. These molecules stimulate leukocyte movement and may explainleukocyte trafficking in different inflammatory situations. Chemokinesmediate the expression of particular adhesion molecules on endothelialcells, and they produce chemoattractants which activate specific celltypes. In addition, the chemokines stimulate proliferation and regulateactivation of specific cell types. In both of these activities,chemokines demonstrate a high degree of target cell specificity.

The chemokine family is divided into two subfamilies based on whethertwo amino terminal cysteine residues are immediately adjacent (C-C) orseparated by one amino acid (C-X-C). Chemokines of the C-X-C familygenerally activate neutrophils and fibroblasts while the C-C chemokinesact on a more diverse group of target cells includingmonocytes/macrophages, basophils, eosinophils and T lymphocytes. Theknown chemokines of both subfamilies are synthesized by many diversecell types as reviewed in Thomson A. (1994) The Cytokine Handbook, 2 dEd. Academic Press, N.Y. Chemokines are also reviewed in Schall T J(1994) Chemotactic Cytokines: Targets for Therapeutic Development.International Business Communications, Southborough Mass. pp 180–270;and in Paul W E (1993) Fundamental Immunology, 3rd Ed. Raven Press, N.Y.pp 822–826.

Known chemokines of the C-X-C subfamily include macrophage inflammatoryproteins alpha and beta (MIP-1 and MIP-2), interleukin-8 (IL-8), andgrowth regulated protein (GRO-alpha and beta).

MIP-2 was first identified as a 6 kDa heparin binding protein secretedby the mouse macrophage cell line RAW 264.7 upon stimulation withlipopolysaccharide (LPS). MIP-2 is a member of the C-X-C (or CXC)subfamily of chemokines. Mouse MIP-2 is chemotactic for humanneutrophils and induces local neutrophil infiltration when injected intothe foot pads of mice. Rat MIP-2 shows 86% amino acid homology to themouse MIP-2 and is chemotactic for rat neutrophils but does notstimulate migration of rat alveolar macrophages or human peripheralblood eosinophils or lymphocytes. In addition, the rat MIP-2 has beenshown to stimulate proliferation of rat alveolar epithelial cells butnot fibroblasts.

Current techniques for diagnosis of abnormalities in inflamed ordiseased issues mainly rely on observation of clinical symptoms orserological analyses of body tissues or fluids for hormones,polypeptides or various metabolites. Problems exist with thesediagnostic techniques. First, patients may not manifest clinicalsymptoms at early stages of disease. Second, serological tests do notalways differentiate between invasive diseases and genetic syndromes.Thus, the identification of expressed chemokines is important to thedevelopment of new diagnostic techniques, effective therapies, and toaid in the understanding of molecular pathogenesis.

To date, chemokines have been implicated in at least the followingconditions: psoriasis, inflammatory bowel disease, renal disease,arthritis, immune-mediated alopecia, stroke, encephalitis, MS,hepatitis, and others. In addition, non-ELR-containing chemokines havebeen implicated in the inhibition of angiogenesis, thus indicating thatthese chemokines have a rule in tumor vascularization and tumorigenesis.

Therefore it is the object of this invention to identify polypeptidesand nucleic acids encoding the same which have sequence identity andsimilarity with cytokine-induced neutrophil chemoattractants, MIP-1,MIP-2, and other related proteins. The efforts of this object areprovided herein.

56. PRO701

Beta neurexins and neuroligins are plasma membrane proteins that aredisplayed on the neuronal cell surface. Neuroligin 1 is enriched insynaptic plasma membranes and acts as a splice site-specific ligand forbeta neurexins as described in Ichtchenko, et al., Cell, 81(3):435–443(1995). The extracellular sequence of neuroligin 1 is composed of acatalytically inactive esterase domain homologous toacetylcholinesterase. Neuroligin 2 and 3 are similar in structure andsequence to neuroligin 1. All neuroligins contain an N-terminalhydrophobic sequence with the characteristics of a cleaved signalpeptide followed by a large esterase homology domain, a highly conservedsingle transmembrane region, and a short cytoplasmic domain. The threeneuroligins are alternatively spliced at the same position and areexpressed at high levels only in the brain. Tight binding of the threeneuroligins to beta neurexins is observed only for beta neurexinslacking an insert in splice site 4. Thus, neuroligins constitute amultigene family of brain-specific proteins with distinct isoforms thatmay have overlapping functions in mediating recognition processesbetween neurons, see Ichtchenko, et al., J. Biol. Chem.,271(5):2676–2682 (1996). Moreover, neurexins and neuroligins have beenreported as functioning as adhesion molecules in a Ca²⁺ dependentreaction that is regulated by alternative splicing of beta neurexins,i.e., see Nguyen and Sudhof, J. Biol. Chem., 272(41):26032–26039 (1997).Given the foregoing, membrane bound proteins are of interest. Moregenerally, membrane-bound proteins and receptors can play an importantrole in the formation, differentiation and maintenance of multicellularorganisms. The fate of many individual cells, e.g., proliferation,migration, differentiation, or interaction with other cells, istypically governed by information received from other cells and/or theimmediate environment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. Such membrane-bound proteins and cellreceptors include, but are not limited to, cytokine receptors, receptorkinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native membrane-bound receptor proteins, particularly those havingsequence identity and/or similarity with neuroligins 1, 2 and 3. Manyefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel secreted andmembrane-bound receptor proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No.5,536,637)]. The results of such efforts are provided herein.

57. PRO704

VIP36 is localized to the Golgi apparatus and the cell surface, andbelongs to a family of legume lectin homologues in the animal secretorypathway that might be involved in the trafficking of glycoproteins,glycolipids, or both. It is further believed that VIP36 binds to sugarresidues of glycosphingolipids and/or gycosylphosphatidyl-inositolanchors and might provide a link between the extracellular/luminal faceof glycolipid rafts and the cytoplasmic protein segregation machinery.Further regarding VIP36, it is believed that there is a signal at itsC-terminus that matches an internalization consensus sequence whichconfers its ability to cycle between the plasma membrane and Golgi. See,Fiedler, et al, EMBO J., 13(7):1729–1740 (1994); Fiedler and Simons, J.Cell Sci., 109(1):271–276 (1996); Itin, et al., MBO J., 14(10):2250–2256(1995). It is believed that VIP36 is either the same as or very closelyrelated to the human GP36b protein. VIP36 and/or GP36b are of interest.

More generally, vesicular, cytoplasmic, extracellular and membrane-boundproteins play important roles in the formation, differentiation andmaintenance of multicellular organisms. The fate of many individualcells, e.g., proliferation, migration, differentiation, or interactionwith other cells, is typically governed by information received fromother cells and/or the immediate environment. This information is oftentransmitted by secreted polypeptides (for instance, mitogenic factors,survival factors, cytotoxic factors, differentiation factors,neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Thesesecreted polypeptides or signaling molecules normally pass through thecellular secretory pathway to reach their site of action in theextracellular environment, usually at a membrane-bound receptor protein.

Secreted proteins have various industrial applications, including use aspharmaceuticals, diagnostics, biosensors and bioreactors. In fact, mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane-bound proteins, also have potential as therapeutic ordiagnostic agents. Receptor immunoadhesins, for instance, can beemployed as therapeutic agents to block receptor-ligand interaction.Membrane-bound proteins can also be employed for screening of potentialpeptide or small molecule inhibitors of the relevant receptor/ligandinteraction. Such membrane-bound proteins and cell receptors include,but are not limited to, cytokine receptors, receptor kinases, receptorphosphatases, receptors involved in cell-cell interactions, and cellularadhesin molecules like selectins and integrins. Transduction of signalsthat regulate cell growth and differentiation is regulated in part byphosphorylation of various cellular proteins. Protein tyrosine kinases,enzymes that catalyze that process, can also act as growth factorreceptors. Examples include fibroblast growth factor receptor and nervegrowth factor receptor.

Efforts are being undertaken by both industry and academia to identifynew, native vesicular, cytoplasmic, secreted and membrane-bound receptorproteins, particularly those having sequence identity and/or similaritywith VIP36. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted and membrane-bound receptor proteins. Examples of screeningmethods and techniques are described in the literature [see, forexample, Klein et al., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S.Pat. No. 5,536,637)].

58. PRO706

Acid phophatase proteins are secreted proteins which dephophorylateterminal phosphate groups under acidic pH conditions. Acid phophatasescontain a RHGXRXP amino acid sequence, which is predicted to bemechanistically significant. Acid phosphatases may have importantfunctions in the diagnosis and treatment of human diseases. For example,prostatic acid phosphatase is a secreted protein uniquely expressed inprostatic tissue and prostate cancer. The level of prostatic acidphosphatase is a potential prognostic factor for local and biochemicalcontrol in prostate cancer patients treated with radiotherapy, asdescribed in Lankford et al., Int. J. Radiat. Oncol. Biol. Phys. 38(2):327–333 (1997). Research suggests that a cellular immune response toprostatic acid phosphatase may mediate destructive autoimmuneprostatitis, and that xenogeneic forms of prostatic acid phosphatase mayprove useful for immunotherapy of prostate cancer. See Fong et al., J.Immunol. 169(7): 3113–3117(1997). Seminal prostatic acid phosphataselevels correlate significantly with very low sperm levels (oligospermia)in individuals over 35, see Singh et al., Singapore Med. J. 37(6):598–599 (1996). Thus, prostatic acid phosphatase has been implicated ina variety of human diseases, and may have an important function indiagnosis and therapy of these diseases. A series ofaminobenzylphosphatic acid compounds are highly potent inhibitors ofprostatic acid phosphatase, as described in Beers et al., Bioorg. Med.Chem. 4(10): 1693–1701 (1996).

More generally, extracellular proteins play an important role in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins, particularly those havingsequence identity with prostate acid phosphatase precursor and lysosomalacid phosphatase precursor and in some cases, those having identity withDNA found in fetal heart. Many efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel secreted proteins. Examples of screening methods and techniquesare described in the literature [see, for example, Klein et al., Proc.Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No. 5,536,637)].

59. PRO707

Cadherins are a large family of transmembrane proteins. At leastcadherins 1–13 as well as types B, E, EP, M, N, P and R have beencharacterized. Among the functions cadherins are known for, with someexceptions, cadherins participate in cell aggregation and are associatedwith cell-cell adhesion sites. Cadherins are further described inTanihara, et al., J. Cell Sci., 107(6):1697–1704 (1994) and Tanihara, etal., Cell Adhes. Commun., 2(1):15–26 (1994). Moreover, it has beenreported that some members of the cadherin superfamily are involved ingeneral cell-cell interaction processes including transduction. See,Suzuki, J. Cell Biochem., 61(4):531–542 (1996). Therefore, novel membersof the cadherin superfamily are of interest.

More generally, all novel proteins are of interest, includingmembrane-bound proteins. Membrane-bound proteins and receptors can playan important role in the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins, particularlymembrane bound proteins having identity with cadherins. The results ofsuch efforts are provided herein.

60. PRO322

Proteases are enzymatic proteins which are involved in a large number ofvery important biological processes in mammalian and non-mammalianorganisms. Numerous different protease enzymes from a variety ofdifferent mammalian and non-mammalian organisms have been bothidentified and characterized, including the serine proteases whichexhibit specific activity toward various serine-containing proteins. Themammalian protease enzymes play important roles in biological processessuch as, for example, protein digestion, activation, inactivation, ormodulation of peptide hormone activity, and alteration of the physicalproperties of proteins and enzymes.

Neuropsin is a novel serine protease whose mRNA is expressed in thecentral nervous system. Mouse neuropsin has been cloned, and studieshave shown that it is involved in the hippocampal plasticity. Neuropsinhas also been indicated as associated with extracellular matrixmodifications and cell migrations. See, generally, Chen, et al.,Neurosci., 7(2):5088–5097 (1995) and Chen, et al., J. Histochem.Cytochem., 46:313–320 (1998).

Efforts are being undertaken by both industry and academia to identifynew, native membrane-bound or secreted proteins, particularly thosehaving homology to neuropsin, serine protease, neurosin and trypsinogen.Many efforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel secreted andmembrane-bound receptor proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No.5,536,637)].

61. PRO526

Protein-protein interactions include those involved with receptor andantigen complexes and signaling mechanisms. As more is known about thestructural and functional mechanisms underlying protein-proteininteractions, protein-protein interactions can be more easilymanipulated to regulate the particular result of the protein-proteininteraction. Thus, the underlying mechanisms of protein-proteininteractions are of interest to the scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglobular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415–421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141–174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215–222(1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndrome,Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1):111–116 (July1995), reporting that platelets have leucine rich repeats and Ruoslahti,E. I., et al., WO9110727-A by La Jolla Cancer Research Foundationreporting that decorin binding to transforming growth factorβ hasinvolvement in a treatment for cancer, wound healing and scarring.Related by function to this group of proteins is the insulin like growthfactor (IGF), in that it is useful in wound-healing and associatedtherapies concerned with re-growth of tissue, such as connective tissue,skin and bone; in promoting body growth in humans and animals; and instimulating other growth-related processes. The acid labile subunit(ALS) of IGF is also of interest in that it increases the half-life ofIGF and is part of the IGF complex in vivo. ALS is further described inLeong and Baxter, Mol. Endocrinol., 6(6):870–876 (1992); Baxter, J.Biol. Chem., 264(20):11843–11848 (1989); and Khosravi, et al., J. Clin.Endocrinol. Metab., 82(12):3944–3951 (1997).

Another protein which has been reported to have leucine-rich repeats isthe SLIT protein which has been reported to be useful in treatingneuro-degenerative diseases such as Alzheimer's disease, nerve damagesuch as in Parkinson's disease, and for diagnosis of cancer, see,Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1 by YaleUniversity. Also of interest is LIG-1, a membrane glycoprotein that isexpressed specifically in glial cells in the mouse brain, and hasleucine rich repeats and immunoglobulin-like domains. Suzuki, et al., J.Biol. Chem. (U.S.), 271(37):22522 (1996). Other studies reporting on thebiological functions of proteins having leucine rich repeats include:Tayar, N., et al., Mol. Cell Endocrinol., (Ireland), 125(1–2):65–70(December 1996) (gonadotropin receptor involvement); Miura, Y., et al.,Nippon Rinsho (Japan), 54(7):1784–1789 (July 1996) (apoptosisinvolvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,6(4):1125–1133 (October 1995) (kidney disease involvement).

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions. Of particular interest are those proteinshaving leucine rich repeats and identity or similarity to known proteinshaving leucine rich repeats such as ALS. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted and membrane-bound proteins having leucinerich repeats. Examples of screening methods and techniques are describedin the literature [see, for example, Klein et al., Proc. Natl. Acad.Sci., 93:7108–7113 (1996); U.S. Pat. No. 5,536,637)].

62. PRO531

Cadherins are a large family of transmembrane proteins. Cadherinscomprise a family of calcium-dependent glycoproteins that function inmediating cell-cell adhesion in virtually all solid tissues ofmulticellular organisms. At least cadherins 1–13 as well as types B, E,EP, M, N, P and R have been characterized. Among the functions cadherinsare known for, with some exceptions, cadherins participate in cellaggregation and are associated with cell-cell adhesion sites. Recently,it has been reported that while all cadherins share multiple repeats ofa cadherin specific motif believed to correspond to folding ofextracellular domains, members of the cadherin superfamily havedivergent structures and, possibly, functions. In particular it has beenreported that members of the cadherin superfamily are involved in signaltransduction. See, Suzuki, J. Cell Biochem., 61(4):531–542 (1996).Cadherins are further described in Tanihara, et al., J. Cell Sci.,107(6):1697–1704 (1994), Aberle, et al., J. Cell Biochem., 61(4):514–523(1996) and Tanihara, et al., Cell Adhes. Commun., 2(1):15–26 (1994).

Protocadherins are members of the cadherin superfamily which are highlyexpressed in the brain. In some studies, protocadherins have shown celladhesion activity. See, Sano, et al., EMBO J., 12(6):2249–2256 (1993).However, studies have also shown that some protocadherins, such asprotocadherin 3 (also referred to as Pcdh3 or pc3), do not show strongcalcium dependent cell aggregation activity. See, Sago, et al.,Genomics, 29(3):631–640 (1995) for this study and furthercharacteristics of Pcdh3.

Therefore, novel members of the cadherin superfamily are of interest.More generally, all membrane-bound proteins and receptors are ofinterest. Such proteins can play an important role in the formation,differentiation and maintenance of multicellular organisms. The fate ofmany individual cells, e.g., proliferation, migration, differentiation,or interaction with other cells, is typically governed by informationreceived from other cells and/or the immediate environment. Thisinformation is often transmitted by secreted polypeptides (for instance,mitogenic factors, survival factors, cytotoxic factors, differentiationfactors, neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Suchmembrane-bound proteins and cell receptors include, but are not limitedto, cytokine receptors, receptor kinases, receptor phosphatases,receptors involved in cell-cell interactions, and cellular adhesinmolecules like selectins and integrins. For instance, transduction ofsignals that regulate cell growth and differentiation is regulated inpart by phosphorylation of various cellular proteins. Protein tyrosinekinases, enzymes that catalyze that process, can also act as growthfactor receptors. Examples include fibroblast growth factor receptor andnerve growth factor receptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are therefore being undertaken by both industry and academia toidentify new, native membrane bound proteins, particular those havingsequence identity with protocadherins, especially 3 and 4. Many effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel membrane-bound proteins.Provided herein are the results of such efforts.

63. PRO534

Protein disulfide isomerase is an enzymatic protein which is involved inthe promotion of correct refolding of proteins through the establishmentof correct disulfide bond formation. Protein disulfide isomerase wasinitially identified based upon its ability to catalyze the renaturationof reduced denatured RNAse (Goldberger et al., J. Biol. Chem.239:1406–1410 (1964) and Epstein et al., Cold Spring Harbor Symp. Quant.Biol. 28:439–449 (1963)). Protein disulfide isomerase has been shown tobe a resident enzyme of the endoplasmic reticulum which is retained inthe endoplasmic reticulum via a -KDEL or -HDEL amino acid sequence atits C-terminus. Protein disulfide isomerase and related proteins arefurther described in Laboissiere, et al., J. Biol. Chem.,270(47:28006–28009 (1995); Jeenes, et al., Gene, 193(2):151–156 (1997;Koivunen, et al., Genomics, 42(3):397–404 (1997); and Desilva, et al.,DNA Cell Biol., 15(1):9–16 (1996). These studies indicate the importanceof the identification of protein disulfide related proteins.

More generally, and also of interest are all novel membrane-boundproteins and receptors. Such proteins can play an important role in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. Such membrane-bound proteins and cellreceptors include, but are not limited to, cytokine receptors, receptorkinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Given the importance of membrane bound proteins, efforts are under wayto identity novel membrane bound proteins. Moreover, given theimportance of disulfide bond-forming enzymes and their potential uses ina number of different applications, for example in increasing the yieldof correct refolding of recombinantly produced proteins, efforts arecurrently being undertaken by both industry and academia to identifynew, native proteins having sequence identity with protein disulfideisomerase. Many of these efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel protein disulfide isomerase homologs. We herein describe a novelpolypeptide having sequence identity with protein disulfide isomeraseand the nucleic acids encoding the same.

64. PRO697

Secreted frizzled related proteins (sFRPs) are related to the frizzledfamily of transmembrane receptors. The sFRPs are approximately 30 kDa insize, and each contains a putative signal sequence, a frizzled-likecysteine-rich domain, and a conserved hydrophilic carboxy-terminaldomain. It has been reported that sFRPs may function to modulate Wntsignaling, or function as ligands for certain receptors. Rattner, etal., PNAS USA, 94(7):2859–2863 (1997). Therefore, sFRPs and proteinshaving sequence identity and/or similarity to sFRPs are of interest.

Another secreted protein of interest is any member of the family ofsecreted apoptosis-related proteins (SARPs). Expression of SARPsmodifies the intracellular levels of beta-catenin, suggesting that SARPsinterfere with the Wnt-frizzled proteins signaling pathway. Melkonyan,et al., PNAS USA, 94(25):13636–13641 (1997). Therefore, SARPs andproteins having sequence identity and/or similarity to SARPs are ofinterest.

In addition to sFRPs and SARPs, many extracellular proteins are ofinterest. Extracellular proteins play an important role in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents.

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins, particularly those having sequenceidentity or similarity with sFRP-2 and SARP-1. Many efforts are focusedon the screening of mammalian recombinant DNA libraries to identify thecoding sequences for novel secreted proteins. Examples of screeningmethods and techniques are described in the literature [see, forexample, Klein et al., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S.Pat. No. 5,536,637)].

65. PRO717

Efforts are being undertaken by both industry and academia to identifynew, native transmembrane receptor proteins. Many efforts are focused onthe screening of mammalian recombinant DNA libraries to identify thecoding sequences for novel receptor proteins. The results of suchefforts are provided herein.

66. PRO731

Cadherins are a large family of transmembrane proteins. Cadherinscomprise a family of calcium-dependent glycoproteins that function inmediating cell-cell adhesion in virtually all solid tissues ofmulticellular organisms. At least cadherins 1–13 as well as types B, E,EP, M, N, P and R have been characterized. Among the functions cadherinsare known for, with some exceptions, cadherins participate in cellaggregation and are associated with cell-cell adhesion sites. Recently,it has been reported that while all cadherins share multiple repeats ofa cadherin specific motif believed to correspond to folding ofextracellular domains, members of the cadherin superfamily havedivergent structures and, possibly, functions. In particular it has beenreported that members of the cadherin superfamily are involved in signaltransduction. See, Suzuki, J. Cell Biochem., 61(4):531–542 (1996).Cadherins are further described in Tanihara, et al., J. Cell Sci.,107(6):1697–1704 (1994), Aberle, et al., J. Cell Biochem., 61(4):514–523(1996) and Tanihara, et al., Cell Adhes. Commun., 2(1):15–26 (1994).

Protocadherins are members of the cadherin superfamily which are highlyexpressed in the brain. In some studies, protocadherins have shown celladhesion activity. See, Sano, et al., EMBO J., 12(6):2249–2256 (1993).However, studies have also shown that some protocadherins, such asprotocadherin 3 (also referred to as Pcdh3 or pc3), do not show strongcalcium dependent cell aggregation activity. See, Sago, et al.,Genomics, 29(3):631–640 (1995) for this study and furthercharacteristics of Pcdh3.

Therefore, novel members of the cadherin superfamily are of interest.More generally, all membrane-bound proteins and receptors are ofinterest. Such proteins can play an important role in the formation,differentiation and maintenance of multicellular organisms. The fate ofmany individual cells, e.g., proliferation, migration, differentiation,or interaction with other cells, is typically governed by informationreceived from other cells and/or the immediate environment. Thisinformation is often transmitted by secreted polypeptides (for instance,mitogenic factors, survival factors, cytotoxic factors, differentiationfactors, neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Suchmembrane-bound proteins and cell receptors include, but are not limitedto, cytokine receptors, receptor kinases, receptor phosphatases,receptors involved in cell-cell interactions, and cellular adhesinmolecules like selectins and integrins. For instance, transduction ofsignals that regulate cell growth and differentiation is regulated inpart by phosphorylation of various cellular proteins. Protein tyrosinekinases, enzymes that catalyze that process, can also act as growthfactor receptors. Examples include fibroblast growth factor receptor andnerve growth factor receptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are therefore being undertaken by both industry and academia toidentify new, native membrane bound proteins, particular those havingsequence identity with protocadherins, especially 4, 68, 43, 42, 3 and5. Many efforts are focused on the screening of mammalian recombinantDNA libraries to identify the coding sequences for novel membrane-boundproteins. Provided herein are the results of such efforts.

67. PRO218

Efforts are being undertaken by both industry and academia to identifynew, native membrane bound proteins, particularly those having sequenceidentity with membrane regulator proteins. Many efforts are focused onthe screening of mammalian recombinant DNA libraries to identify thecoding sequences for novel receptor proteins.

68. PRO768

The integrins comprise a supergene family of cell-surface glycoproteinreceptors that promote cellular adhesion. Each cell has numerousreceptors that define its cell adhesive capabilities. Integrins areinvolved in a wide variety of interaction between cells and other cellsor matrix components. The integrins are of particular importance inregulating movement and function of immune system cells. The plateletIIb/IIIA integrin complex is of particular importance in regulatingplatelet aggregation. A member of the integrin family, integrin β-6, isexpressed on epithelial cells and modulates epithelial inflammation.Another integrin, leucocyte-associated antigen-1 (LFA-1) is important inthe adhesion of lymphocytes during an immune response.

Of particular interest is H36-alpha 7, an integrin alpha chain that isdevelopmentally regulated during myogenesis as described in Song, etal., J. Cell Biol., 117(3):643–657 (1992). The expression pattern of thelaminin-binding alpha 7 beta 1 integrin is developmentally regulated inskeletal, cardiac, and smooth muscle. Ziober, et al., Mol. Biol. Cell,8(9): 1723–1734 (1997). It has been reported that expression of thealpha 7-X1/X2 integrin is a novel mechanism that regulates receptoraffinity states in a cell-specific context and may modulateintegrin-dependent events during muscle development and repair. Id. Ithas further been reported that laminins promote the locomotion ofskeletal myoblasts via the alpha 7 integrin receptor. In particular itwas reported that alpha 7 beta 1 receptor can promote myoblast adhesionand motility on a restricted number of laminin isoforms and may beimportant in myogenic precursor recruitment during regeneration anddifferentiation. Yao, et al., J. Cell Sci., 109(13):3139–3150 (1996).Spliced variants of integrin alpha 7 are also described in Leung, etal., Biochem. Biophys. Res. Commun., 243(1):317–325 (1998) and Fornaroand Languino, Matrix Biol., 16(4):185–193 (1997). Moreover, it has beenreported that absence of integrin alpha 7 causes a form of musculardystrophy. Thus integrins, particularly those related to integrin 7 andrelated molecules, are of interest.

In addition to the interest of integrins, more generally, allmembrane-bound proteins and receptors are of interest since suchproteins can play an important role in the formation, differentiationand maintenance of multicellular organisms. The fate of many individualcells, e.g., proliferation, migration, differentiation, or interactionwith other cells, is typically governed by information received fromother cells and/or the immediate environment. This information is oftentransmitted by secreted polypeptides (for instance, mitogenic factors,survival factors, cytotoxic factors, differentiation factors,neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Suchmembrane-bound proteins and cell receptors include, but are not limitedto, cytokine receptors, receptor kinases, receptor phosphatases,receptors involved in cell-cell interactions, and cellular adhesinmolecules like selectins and integrins. For instance, transduction ofsignals that regulate cell growth and differentiation is regulated inpart by phosphorylation of various cellular proteins. Protein tyrosinekinases, enzymes that catalyze that process, can also act as growthfactor receptors. Examples include fibroblast growth factor receptor andnerve growth factor receptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Therefore, efforts are being undertaken by both industry and academia toidentify new, native receptor proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel receptor proteins. The results of such efforts,particularly those focused on identifying new polypeptides havingsequence identity with integrins, are provided herein.

69. PRO771

Testican is a multidomain testicular proteoglycan which is expressed innumerous tissue types including, but not limited to neuromusculartissue, the brain and reproductive tissues. Testican resemblesmodulators of cell social behavior such as the regulation of cell shape,adhesion, migration and proliferation. [Bonnet, F. et al., J. Biol.Chem., 271(8):4373 (1996), Perin, J. P. et al., EXS (Switzerland),70:191 (1994), Alliel, P. M., et al, Eur. J. Biochem., 214(1):346(1993), Charbonnier, F., et al., C. R. Seances Soc. Biol. Fil. (France),191(1):127 (1997)]. Among other reasons, since testican has beenimplicated in neuronal processes and may be associated with the growthof connective tissue, testican and related molecules are of interest.

More generally, all extracellular proteins are of interest.Extracellular proteins play an important role in the formation,differentiation and maintenance of multicellular organisms. The fate ofmany individual cells, e.g., proliferation, migration, differentiation,or interaction with other cells, is typically governed by informationreceived from other cells and/or the immediate environment. Thisinformation is often transmitted by secreted polypeptides (for instance,mitogenic factors, survival factors, cytotoxic factors, differentiationfactors, neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Thesesecreted polypeptides or signaling molecules normally pass through thecellular secretory pathway to reach their site of action in theextracellular environment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No.5,536,637)]. The results of such efforts, particularly those focused onidentifying molecules having identity and/or similarity with testicanare of interest.

70. PRO733

T1/ST2 is a receptor-like molecule homologous to the type Iinterleukin-1 receptor, believed to be involved in cell signaling. TheT1/ST2 receptor and/or putative ligands are further described in Gayle,et al., J. Biol. Chem., 271(10):5784–5789 (1996), Kumar, et al., J.Biol. Chem., 270(46):27905–27913 (1995), and Mitcham, et al., J. Biol.Chem., 271(10):5777–5783 (1996). These proteins, and proteins relatedthereto are of interest.

More generally all membrane-bound proteins and receptors are of interestsince they can play an important role in the formation, differentiationand maintenance of multicellular organisms. The fate of many individualcells, e.g., proliferation, migration, differentiation, or interactionwith other cells, is typically governed by information received fromother cells and/or the immediate environment. This information is oftentransmitted by secreted polypeptides (for instance, mitogenic factors,survival factors, cytotoxic factors, differentiation factors,neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Suchmembrane-bound proteins and cell receptors include, but are not limitedto, cytokine receptors, receptor kinases, receptor phosphatases,receptors involved in cell-cell interactions, and cellular adhesinmolecules like selectins and integrins. For instance, transduction ofsignals that regulate cell growth and differentiation is regulated inpart by phosphorylation of various cellular proteins. Protein tyrosinekinases, enzymes that catalyze that process, can also act as growthfactor receptors. Examples include fibroblast growth factor receptor andnerve growth factor receptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel receptor proteins. The results of such efforts are providedherein.

71. PRO162

Pancreatitis-associated protein (PAP) is a secretory protein that isoverexpressed by the pancreas during acute pancreatitis. Serum PAPconcentrations have been shown to be abnormally high in patients withacute pancreatitis. Pezzilli et al., Am. J. Gastroenterol.,92(10):1887–1890 (1997).

PAP is synthesized by the pancreas due to pancreatic inflammation andhas been shown to be a good serum marker for injury of the pancreas. Inaddition, serum PAP levels appear to strongly correlate with creatinineclearance measurements. In patients with a pancreas-kidneytransplantation, PAP may prove to be a useful biological andhistological marker of pancreatic graft rejection. Van der Pijl et al.,Transplantation, 63(7):995–1003 (1997). Further, PAP has been shown tobe useful in screening neonates for cystic fibrosis. In fact, PAP maydiscriminate cystic fibrosis neonates with better specificity than thecurrent immunoreactive trypsis assay. Iovanna et al., C. R. Acad. Aci.III, 317(6):561–564.

Secreted proteins such as PAP have various industrial applications,including pharmaceuticals, diagnostics, biosensors and bioreactors. Mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane proteins, also have potential as therapeutic ordiagnostic agents.

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No.5,536,637)]. The results of such efforts are presented herein.

72. PRO788

Anti-neoplastic urinary protein (ANUP) was identified as the majorprotein present in a fraction of human urine which exhibitsantiproliferative activity against human tumor cell lines withoutaffecting the growth of several normal diploid cell lines or tumor cellsof mouse or hamster origin. Sloane et al., Biochem. J., 234(2):355–362(1986).

ANUP is a unique cytokine that has been found in human granulocytes. TheN-terminal amino acid sequence has been shown to be unique. A syntheticpeptide corresponding to the first nine residues, with Cys at positions4 and 7, was found to be an anti-tumor agent in vitro. Ridge and Sloane,Cytokine, 8(1):1–5 (1996).

Secreted proteins such as ANUP have various industrial applications,including pharmaceuticals, diagnostics, biosensors and bioreactors. Mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane proteins, also have potential as therapeutic ordiagnostic agents. Efforts are being undertaken by both industry andacademia to identify new, native secreted proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted proteins. Examples ofscreening methods and techniques are described in the literature [see,for example, Klein et al., Proc. Natl. Acad. Sci., 93:7108–7113 (1996);U.S. Pat. No. 5,536,637)].

73. PRO1008

Dickkopf-1 (dkk-1) is a member of a family of secreted proteins andfunctions in head induction. Dkk-1 is an inducer of Spemann organizer inamphibian embryos. Glinka, et al., Nature, 391(6665):357–362 (1998).Dkk-1 is a potent antagonist of Wnt signalling, suggesting that dkkgenes encode a family of secreted Wnt inhibitors. Thus, dkk-1 familymembers and related molecules are of interest.

More generally, all extracellular proteins are of interest since theycan play an important role in the formation, differentiation andmaintenance of multicellular organisms. The fate of many individualcells, e.g., proliferation, migration, differentiation, or interactionwith other cells, is typically governed by information received fromother cells and/or the immediate environment. This information is oftentransmitted by secreted polypeptides (for instance, mitogenic factors,survival factors, cytotoxic factors, differentiation factors,neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Thesesecreted polypeptides or signaling molecules normally pass through thecellular secretory pathway to reach their site of action in theextracellular environment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents.

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins, particularly those related to dkk-1. Manyefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel secreted proteins.Examples of screening methods and techniques are described in theliterature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108–7113 (1996); U.S. Pat. No. 5,536,637)]. The results of suchefforts to identify molecules related to dkk-1 are provided herein.

74. PRO1012

Protein disulfide isomerase is an enzymatic protein which is involved inthe promotion of correct refolding of proteins through the establishmentof correct disulfide bond formation. Protein disulfide isomerase wasinitially identified based upon its ability to catalyze the renaturationof reduced denatured RNAse (Goldberger et al., J. Biol. Chem.239:1406–1410 (1964) and Epstein et al., Cold Spring Harbor Symp. Quant.Biol. 28:439–449 (1963)). Protein disulfide isomerase has been shown tobe a resident enzyme of the endoplasmic reticulum which is retained inthe endoplasmic reticulum via a -KDEL or -HDEL amino acid sequence atits C-terminus. Protein disulfide isomerase and related proteins arefurther described in Laboissiere, et al., J. Biol. Chem.,270(47:28006–28009 (1995); Jeenes, et al., Gene, 193(2):151–156 (1997;Koivunen, et al., Genomics, 42(3):397–404 (1997); and Desilva, et al.,DNA Cell Biol., 15(1):9–16 (1996). These studies indicate the importanceof the identification of protein disulfide related proteins.

More generally, the identification of all extracellular andmembrane-bound proteins is of interest since they play important rolesin the formation, differentiation and maintenance of multicellularorganisms. The fate of many individual cells, e.g., proliferation,migration, differentiation, or interaction with other cells, istypically governed by information received from other cells and/or theimmediate environment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment, usually at amembrane-bound receptor protein.

Secreted proteins have various industrial applications, including use aspharmaceuticals, diagnostics, biosensors and bioreactors. In fact, mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane-bound proteins, also have potential as therapeutic ordiagnostic agents. Receptor immunoadhesins, for instance, can beemployed as therapeutic agents to block receptor-ligand interaction.Membrane-bound proteins can also be employed for screening of potentialpeptide or small molecule inhibitors of the relevant receptor/ligandinteraction. Such membrane-bound proteins and cell receptors include,but are not limited to, cytokine receptors, receptor kinases, receptorphosphatases, receptors involved in cell-cell interactions, and cellularadhesin molecules like selectins and integrins. Transduction of signalsthat regulate cell growth and differentiation is regulated in part byphosphorylation of various cellular proteins. Protein tyrosine kinases,enzymes that catalyze that process, can also act as growth factorreceptors. Examples include fibroblast growth factor receptor and nervegrowth factor receptor.

Of particular interest are cellular proteins having endoplasmicreticulum (ER) retention signals. These proteins are retained in thecell and function closely with endoplasmic reticulum in proteinproduction. Such proteins have been described previously, i.e., seeShorrosh and Dixon, Plant J., 2(1):51–58 (1992).

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins, and inparticular, cellular proteins having ER retension signals. Many effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No. 5,536,637)]. The resultsof such efforts, particularly the identification of novel polypeptidesand nucleic acids encoding the same, which have sequence identity andsimilarity to protein disulfide isomerase are presented herein.

75. PRO1014

Oxygen free radicals and antioxidants appear to play an important rolein the central nervous system after cerebral ischemia and reperfusion.Moreover, cardiac injury, related to ischaemia and reperfusion has beenreported to be caused by the action of free radicals. Additionally,studies have reported that the redox state of the cell is a pivotaldeterminant of the fate of the cells. Furthermore, reactive oxygenspecies have been reported to be cytotoxic, causing inflammatorydisease, including tissue necrosis, organ failure, atherosclerosis,infertility, birth defects, premature aging, mutations and malignancy.Thus, the control of oxidation and reduction is important for a numberof reasons including for control and prevention of strokes, heartattacks, oxidative stress and hypertension. In this regard, reductases,and particularly, oxidoreductases, are of interest. Publications furtherdescribing this subject matter include Kelsey, et al., Br. J. Cancer,76(7):85–24 (1997); Friedrich and Weiss, J. Theor. Biol., 187(4):529–40(1997) and Pieulle, et al., J. Bacteriol., 179(18):5684–92 (1997).

In addition to reductases in particular, novel polypeptides aregenerally of interest. Extracellular proteins play an important role inthe formation, differentiation and maintenance of multicellularorganisms. The fate of many individual cells, e.g., proliferation,migration, differentiation, or interaction with other cells, istypically governed by information received from other cells and/or theimmediate environment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No.5,536,637)]. The results of such efforts, particularly those identifyingpolypeptides having sequence identity with reductases, and the nucleicacids encoding the same, are presented herein.

76. PRO1017

Enzymatic proteins play important roles in the chemical reactionsinvolved in the digestion of foods, the biosynthesis of macromolecules,the controlled release and utilization of chemical energy, and otherprocesses necessary to sustain life. Sulfotransferases are enzymes whichtransfer sulfate from a sulfate donor to acceptor substrates,particularly those containing terminal glucoronic acid. The HNK-1carbohydrate epitope is expressed on several neural adhesionglycoproteins and a glycolipid, and is involved in cell interactions.The glucuronyltransferase and sulfotransferase are considered to be thekey enzymes in the biosynthesis of this epitope because the rest of thestructure occurs often in glycoconjugates. HNK-1 sulfotransfererase isfurther described in Bakker, H., et al., J. Biol. Chem.,272(47):29942–29946 (1997).

In addition to HNK-1 sulfotransfererase, and novel proteins relatedthereto, all novel proteins are of interest. Extracellular andmembrane-bound proteins play important roles in the formation,differentiation and maintenance of multicellular organisms. The fate ofmany individual cells, e.g., proliferation, migration, differentiation,or interaction with other cells, is typically governed by informationreceived from other cells and/or the immediate environment. Thisinformation is often transmitted by secreted polypeptides (for instance,mitogenic factors, survival factors, cytotoxic factors, differentiationfactors, neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Thesesecreted polypeptides or signaling molecules normally pass through thecellular secretory pathway to reach their site of action in theextracellular environment, usually at a membrane-bound receptor protein.

Secreted proteins have various industrial applications, including use aspharmaceuticals, diagnostics, biosensors and bioreactors. In fact, mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane-bound proteins, also have potential as therapeutic ordiagnostic agents. Receptor immunoadhesins, for instance, can beemployed as therapeutic agents to block receptor-ligand interaction.Membrane-bound proteins can also be employed for screening of potentialpeptide or small molecule inhibitors of the relevant receptor/ligandinteraction. Such membrane-bound proteins and cell receptors include,but are not limited to, cytokine receptors, receptor kinases, receptorphosphatases, receptors involved in cell-cell interactions, and cellularadhesin molecules like selectins and integrins. Transduction of signalsthat regulate cell growth and differentiation is regulated in part byphosphorylation of various cellular proteins. Protein tyrosine kinases,enzymes that catalyze that process, can also act as growth factorreceptors. Examples include fibroblast growth factor receptor and nervegrowth factor receptor.

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins, particularlythose having sequence identity with HNK-1 sulfotransferase. Many effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108–7113 (1996); U.S. Pat. No. 5,536,637)]. The resultsof such efforts are provided herein.

77. PRO474

Enzymatic proteins play important roles in the chemical reactionsinvolved in the digestion of foods, the biosynthesis of macromolecules,the controlled release and utilization of chemical energy, and otherprocesses necessary to sustain life. Glucose dehydrogenase functions inthe oxidation of glucose to gluconate to generate metabolically usefulenergy. The regulation of the PQQ-linked glucose dehydrogenase indifferent organisms is reviewed in Neijssel, et al., Antonie VanLeeuwenhoek, 56(1):51–61 (1989). Glucose dehydrogenase functions as anauxiliary energy generating mechanism, because it is maximallysynthesized under conditions of energy stress. In addition to moleculesrelated to glucose dehydrogenase, all novel proteins are of interest.Extracellular and membrane-bound proteins play important roles in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment, usually at amembrane-bound receptor protein.

Secreted proteins have various industrial applications, including use aspharmaceuticals, diagnostics, biosensors and bioreactors. In fact, mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane-bound proteins, also have potential as therapeutic ordiagnostic agents. Receptor immunoadhesins, for instance, can beemployed as therapeutic agents to block receptor-ligand interaction.Membrane-bound proteins can also be employed for screening of potentialpeptide or small molecule inhibitors of the relevant receptor/ligandinteraction. Such membrane-bound proteins and cell receptors include,but are not limited to, cytokine receptors, receptor kinases, receptorphosphatases, receptors involved in cell-cell interactions, and cellularadhesin molecules like selectins and integrins. Transduction of signalsthat regulate cell growth and differentiation is regulated in part byphosphorylation of various cellular proteins. Protein tyrosine kinases,enzymes that catalyze that process, can also act as growth factorreceptors. Examples include fibroblast growth factor receptor and nervegrowth factor receptor.

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins, andparticularly cellular proteins and those related to dehydrogenase oroxidoreductase. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted and membrane-bound receptor proteins. Examples of screeningmethods and techniques are described in the literature [see, forexample, Klein et al., Proc. Natl. Acad. Sci., 93:7108–7113 (1996); U.S.Pat. No. 5,536,637)]. The results of such efforts are presented herein.

78. PRO1031

It has been reported that the cytokine interleukin 17 (IL-17) stimulatesepithelial, endothelial, and fibroblastic cells to secrete cytokinessuch as IL1, IL-8, and granulocyte-colony-stimulating factor, as well asprostaglandin E2. Moreover, it has been shown that when cultured in thepresence of IL-17, fibroblasts could sustain proliferation of CD34+preferential maturation into neutrophils. Thus it has been suggestedthat IL-17 constitutes an early initiator of the T cell-dependentinflammatory reaction and/or an element of the cytokine network thatbridges the immune system to hematopoiesis. See, Yao, et al., J.Immunol., 155(12):5483–5486 (1995); Fossiez, et al., J. Exp. Med.,183(6):2593–2603 (1996); Kennedy, et al., J. Interferon Cytokine Res.,16(8):611–617 (1996). Thus, proteins related to IL-17 are of interest.

More generally, all novel proteins are of interest. Extracellularproteins play an important role in the formation, differentiation andmaintenance of multicellular organisms. The fate of many individualcells, e.g., proliferation, migration, differentiation, or interactionwith other cells, is typically governed by information received fromother cells and/or the immediate environment. This information is oftentransmitted by secreted polypeptides (for instance, mitogenic factors,survival factors, cytotoxic factors, differentiation factors,neuropeptides, and hormones) which are, in turn, received andinterpreted by diverse cell receptors or membrane-bound proteins. Thesesecreted polypeptides or signaling molecules normally pass through thecellular secretory pathway to reach their site of action in theextracellular environment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents.

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins, particularly those related to IL-17. Manyefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel secreted proteins.Examples of screening methods and techniques are described in theliterature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108–7113 (1996); U.S. Pat. No. 5,536,637)]. The results of suchefforts are presented herein.

79. PRO938

Protein disulfide isomerase is an enzymatic protein which is involved inthe promotion of correct refolding of proteins through the establishmentof correct disulfide bond formation. Protein disulfide isomerase wasinitially identified based upon its ability to catalyze the renaturationof reduced denatured RNAse (Goldberger et al., J. Biol. Chem.239:1406–1410 (1964) and Epstein et al., Cold Spring Harbor Symp. Quant.Biol. 28:439–449 (1963)). Protein disulfide isomerase has been shown tobe a resident enzyme of the endoplasmic reticulum which is retained inthe endoplasmic reticulum via a -KDEL or -HDEL amino acid sequence atits C-terminus. Protein disulfide isomerase and related proteins arefurther described in Laboissiere, et al., J. Biol. Chem.,270(47):28006–28009 (1995); Jeenes, et al., Gene, 193(2:151–156 (1997);Koivunen, et al., Genomics, 42(3):397–404 (1997); Desilva, et al., DNACell Biol., 15(1):9–16 (1996); Freedman, et al. Trends in Biochem. Sci.19:331–336 (1994); Bulleid, N. J. Advances in Prot. Chem. 44:125–50(1993); and Noiva, R., Prot. Exp. and Purification 5:1–13 (1994). Thesestudies indicate the importance of the identification of proteindisulfide related proteins.

More generally, and also of interest are all novel membrane-boundproteins and receptors. Such proteins can play an important role in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. Such membrane-bound proteins and cellreceptors include, but are not limited to, cytokine receptors, receptorkinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Given the importance of membrane bound proteins, efforts are under wayto identity novel membrane bound proteins. Moreover, given theimportance of disulfide bond-forming enzymes and their potential uses ina number of different applications, for example in increasing the yieldof correct refolding of recombinantly produced proteins, efforts arecurrently being undertaken by both industry and academia to identifynew, native proteins having sequence identity with protein disulfideisomerase. Many of these efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel protein disulfide isomerase homologs.

We herein describe the identification and characterization of a novelpolypeptide having homology to protein disulfide isomerase.

80. PRO1082

The low density lipoprotein (LDL) receptor is a membrane-bound proteinthat plays a key role in cholesterol homeostasis, mediating cellularuptake of lipoprotein particles by high affinity binding to its ligands,apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of theLDL receptor contains 7 cysteine-rich repeats of approximately 40 aminoacids, wherein each repeat contains 6 cysteines, which form 3intra-repeat disulfide bonds. These unique structural features providethe LDL receptor with its ability to specifically interact with apoB-100 and apoE, thereby allowing for transport of these lipoproteinparticles across cellular membranes and metabolism of their components.Soluble fragments containing the extracellular domain of the LDLreceptor have been shown to retain the ability to interact with itsspecific lipoprotein ligands (Simmons et al., J. Biol. Chem.272:25531–25536 (1997)). LDL receptors are further described in Javitt,FASEB J., 9(13):1378–1381 (1995) and Herz and Willnow, Ann. NY Acad.Sci., 737:14–19 (1994). Thus, proteins having sequence identity with LDLreceptors are of interest.

More generally, all membrane-bound proteins and receptors can play animportant role in the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor. Of particular interest are membrane bound proteins that havetype II transmembrane domains.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are thus being undertaken by both industry and academia toidentify new, native proteins, particularly membrane bound proteinsincluding type II transmembrane bound proteins. Many efforts are focusedon the screening of mammalian recombinant DNA libraries to identify thecoding sequences for novel receptor proteins. The results of suchefforts are provided herein.

81. PRO1083

Of particular interest are membrane bound proteins that belong to theseven transmembrane (7TM) receptor superfamily. Examples of thesereceptors include G-protein coupled receptors such as ion receptors.Another example of a 7TM receptor superfamily member is described inOsterhoff, et al., DNA Cell Biol., 16(4):379–389 (1997).

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel receptor proteins. The results of such efforts are presentedherein.

82. PRO200

Polypeptides involved in survival, proliferation and/or differentiationof cells are of interest. Polypeptides known to be involved in thesurvival, proliferation and/or differentiation of cells include VEGF andmembers of the bone morphogenetic protein family. Therefore, novelpolypeptides which are related to either VEGF or the bone morphogeneticprotein are of interest.

The heparin-binding endothelial cell-growth factor, VEGF, was identifiedand purified from media conditioned by bovine pituitary follicular orfolliculo-stellate cells over several years ago. See Ferrara et al.,Biophys. Res. Comm. 161, 851 (1989). VEGF is a naturally occurringcompound that is produced in follicular or folliculo-stellate cells(FC), a morphologically well characterized population of granular cells.The FC are stellate cells that send cytoplasmic processes betweensecretory cells.

VEGF is expressed in a variety of tissues as multiple homodimeric forms(121, 165, 189 and 206 amino acids per monomer) resulting fromalternative RNA splicing. VEGF₁₂₁ is a soluble mitogen that does notbind heparin; the longer forms of VEGF bind heparin with progressivelyhigher affinity. The heparin-binding forms of VEGF can be cleaved in thecarboxy terminus by plasmin to release (a) diffusible form(s) of VEGF.Amino acid sequencing of the carboxy terminal peptide identified afterplasmin cleavage is Arg₁₁₀–Ala₁₁₁. Amino terminal “core” protein, VEGF(1–110) isolated as a homodimer, binds neutralizing monoclonalantibodies (4.6.1 and 2E3) and soluble forms of FMS-like tyrosine kinase(FLT-1), kinase domain region (KDR) and fetal liver kinase (FLK)receptors with similar affinity compared to the intact VEGF₁₆₅homodimer.

As noted, VEGF contains two domains that are responsible respectivelyfor binding to the KDR and FLT-1 receptors. These receptors exist onlyon endothelial (vascular) cells. As cells become depleted in oxygen,because of trauma and the like, VEGF production increases in such cellswhich then bind to the respective receptors in order to signal ultimatebiological effect. The signal then increases vascular permeability andthe cells divide and expand to form new vascular pathways—vasculogenesisand angiogenesis.

Thus, VEGF is useful for treating conditions in which a selected actionon the vascular endothelial cells, in the absence of excessive tissuegrowth, is important, for example, diabetic ulcers and vascular injuriesresulting from trauma such as subcutaneous wounds. Being a vascular(artery and venus) endothelial cell growth factor, VEGF restores cellsthat are damaged, a process referred to as vasculogenesis, andstimulates the formulation of new vessels, a process referred to asangiogenesis.

VEGF would also find use in the restoration of vasculature after amyocardial infarct, as well as other uses that can be deduced. In thisregard, inhibitors of VEGF are sometimes desirable, particularly tomitigate processes such as angiogenesis and vasculogenesis in cancerouscells.

Regarding the bone morphogenetic protein family, members of this familyhave been reported as being involved in the differentiation of cartilageand the promotion of vascularization and osteoinduction in preformedhydroxyapatite. Zou, et al., Genes Dev. (U.S.), 11(17):2191 (1997);Levine, et al., Ann. Plast. Surg., 39(2):158 (1997). A number of relatedbone morphogenetic proteins have been identified, all members of thebone morphogenetic protein (BMP) family. Bone morphogenetic native andmutant proteins, nucleic acids encoding therefor, related compoundsincluding receptors, host cells and uses are further described in atleast: U.S. Pat. Nos. 5,670,338; 5,454,419; 5,661,007; 5,637,480;5,631,142; 5,166,058; 5,620,867; 5,543,394; 4,877,864; 5,013,649;55,106,748; and 5,399,677. Of particular interest are proteins havinghomology with bone morphogenetic protein 1, a procollagen C-proteinasethat plays key roles in regulating matrix deposition.

The present invention is predicated upon research intended to identifynovel polypeptides which are related to VEGF and the BMP family, and inparticular, polypeptides which have a role in the survival,proliferation and/or differentiation of cells. While the novelpolypeptides are not expected to have biological activity identical tothe known polypeptides to which they have homology, the knownpolypeptide biological activities can be used to determine the relativebiological activities of the novel polypeptides. In particular, thenovel polypeptides described herein can be used in assays which areintended to determine the ability of a polypeptide to induce survival,proliferation or differentiation of cells. In turn, the results of theseassays can be used accordingly, for diagnostic and therapeutic purposes.The results of such research is the subject of the present invention.

83. PRO285 and PRO286

The cloning of the Toll gene of Drosophila, a maternal effect gene thatplays a central role in the establishment of the embryonicdorsal-ventral pattern, has been reported by Hashimoto et al., Cell 52,269–279 (1988). The Drosophila Toll gene encodes an integral membraneprotein with an extracytoplasmic domain of 803 amino acids and acytoplasmic domain of 269 amino acids. The extracytoplasmic domain has apotential membrane-spanning segment, and contains multiple copies of aleucine-rich segment, a structural motif found in many transmembraneproteins. The Toll protein controls dorsal-ventral patterning inDrosophila embryos and activates the transcription factor Dorsal uponbinding to its ligand Spätzle. (Morisato and Anderson, Cell 76, 677–688(1994).) In adult Drosophila, the Toll/Dorsal signaling pathwayparticipates in the anti-fungal immune response. (Lenaitre et al., Cell86, 973–983 (1996).)

A human homologue of the Drosophila Toll protein has been described byMedzhitov et al., Nature 388 394–397 (1997). This human Toll, just asDrosophila Toll, is a type I transmembrane protein, with anextracellular domain consisting of 21 tandemly repeated leucine-richmotifs (leucine-rich region—LRR), separated by a non-LRR region, and acytoplasmic domain homologous to the cytoplasmic domain of the humaninterleukin-1 (IL-1) receptor. A constitutively active mutant of thehuman Toll transfected into human cell lines was shown to be able toinduce the activation of NF-κB and the expression of NF-κB-controlledgenes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as theexpression of the constimulatory molecule B7.1, which is required forthe activation of native T cells. It has been suggested that Tollfunctions in vertebrates as a non-clonal receptor of the immune system,which can induce signals for activating both an innate and an adaptiveimmune response in vertebrates. The human Toll gene reported byMedzhitov et al., supra was most strongly expressed in spleen andperipheral blood leukocytes (PBL), and the authors suggested that itsexpression in other tissues may be due to the presence of macrophagesand dendritic cells, in which it could act as an early-warning systemfor infection. The public GenBank database contains the following Tollsequences: Toll (DNAX# HSU88540-1, which is identical with the randomsequenced full-length cDNA #HUMRSC786-1); Toll2 (DNAX# HSU88878-1);Toll3 (DNAX# HSU88879-1); and Toll4 (DNAX# HSU88880-1, which isidentical with the DNA sequence reported by Medzhitov et al., supra). Apartial Toll sequence (Toll5) is available from GenBank under DNAX#HSU88881-1.

Further human homologues of the Drosophila Toll protein, designated asToll-like receptors (huTLRs1–5) were recently cloned and shown to mirrorthe topographic structure of the Drosophila counterpart (Rock et al.,Proc. Natl. Acad. Sci. USA 95, 588–593 [1998]). Overexpression of aconstitutively active mutant of one human TLR (Toll-proteinhomologue—Medzhitov et al., supra; TLR4—Rock et al., supra) leads to theactivation of NF-RκB and induction of the inflammatory cytokines andconstimulatory molecules. Medzhitov et al., supra.

84. PRO213-1, PRO1330 and PRO1449

Cancer is characterized by the increase in the number of abnormal, orneoplastic, cells derived from a normal tissue which proliferate to forma tumor mass, the invasion of adjacent tissues by these neoplastic tumorcells, and the generation of malignant cells which eventually spread viathe blood or lymphatic system to regional lymph nodes and to distantsites (metastasis). In a cancerous state a cell proliferates underconditions in which normal cells would not grow. Cancer manifests itselfin a wide variety of forms, characterized by different degrees ofinvasiveness and aggressiveness.

Alteration of gene expression is intimately related to the uncontrolledcell growth and de-differentiation which are a common feature of allcancers. The genomes of certain well studied tumors have been found toshow decreased expression of recessive genes, usually referred to astumor suppression genes, which would normally function to preventmalignant cell growth, and/or overexpression of certain dominant genes,such as oncogenes, that act to promote malignant growth. Each of thesegenetic changes appears to be responsible for importing some of thetraits that, in aggregate, represent the full neoplastic phenotype(Hunter, Cell 64, 1129 [1991]; Bishop, Cell 64, 235–248 [1991]).

A well known mechanism of gene (e.g. oncogene) overexpression in cancercells is gene amplification. This is a process where in the chromosomeof the ancestral cell multiple copies of a particular gene are produced.The process involves unscheduled replication of the region of chromosomecomprising the gene, followed by recombination of the replicatedsegments back into the chromosome (Alitalo et al., Adv. Cancer Res. 47,235–281 [1986]). It is believed that the overexpression of the geneparallels gene amplification, i.e. is proportionate to the number ofcopies made.

Proto-oncogenes that encode growth factors and growth factor receptorshave been identified to play important roles in the pathogenesis ofvarious human malignancies, including breast cancer. For example, it hasbeen found that the human ErbB2 gene (erbB2, also known as her2, orc-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor(p185HER2; HER2) related to the epidermal growth factor receptor (EGFR),is overexpressed in about 25% to 30% of human breast cancer (Slamon etal., Science 235:177–182 [1987]; Slamon et al., Science 244:707–712[1989]).

It has been reported that gene amplification of a protooncogene is anevent typically involved in the more malignant forms of cancer, andcould act as a predictor of clinical outcome (Schwab et al., GenesChromosomes Cancer 1, 181–193 [1990]; Alitalo et al., supra). Thus,erbB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene 159:19–27 [1995]; and Hynes and Stem, Biochem BiophysActa 1198: 165–184 [1994]), and has been linked to sensitivity and/orresistance to hormone therapy and chemotherapeutic regimens, includingCMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines(Baselga et al., Oncology 11 (3 Suppl 1):43–48 [1997]). However, despitethe association of erbB2 overexpression with poor prognosis, the odds ofHER2-positive patients responding clinically to treatment with taxaneswere greater than three times those of HER2-negative patients (Ibid). Arecombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (ahumanized version of the murine anti-ErbB2 antibody 4D5, referred to asrhuMAb HER2 or Herceptin 7δ) has been clinically active in patients withErbB2-overexpressing metastatic breast cancers that had receivedextensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol.14:737–744 [1996]).

The protein Notch and its homologues are key regulatory receptors indetermining the cell fate in various development processes. The proteinNotch-4, also known as int-3 oncogene, was originally identified as afrequent target in mouse mammary tumor virus (MMVS). Notch-4 is believedto be a transgene which affects the differentiation capacity of stemcells and leads to neoplastic proliferation in epithelial cells.Shirayoshi et al., Genes Cells 2(3):213–224 (1997). Duringembryogenesis, the expression of Notch-4 was detected in endothelialcells of blood vessels forming tissues such as the dorsal aorta,intersegmental vessels, yolk sac vessels, cephalic vessels, heart,vessels in branchial arches, and capillary plexuses. Notch-4 expressionin these tissues was also associated with flk-1, the major regulatorygene of vasculogenesis and angiogenesis. Notch-4 is also upregulated invitro during the differentiation of endothelial stem cell. Theendothelial cell specific expression pattern of Notch-4, as well as itsstructural similarity to Notch suggest that Notch-4 is an endothelialcell specific homologue of Notch and that it may play a role invaculogenesis and angiogenesis.

85. PRO298

Efforts are being undertaken by both industry and academia to identifynew, native receptor proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel receptor proteins. We herein describe the identification andcharacterization of novel transmembrane polypeptides, designated hereinas PRO298 polypeptides.

86. PRO337

Neuronal development in higher vertebrates is characterized by processesthat must successfully navigate distinct cellular environment en routeto their synaptic targets. The result is a functionally preciseformation of neural circuits. The precision is believed to result formmechanisms that regulate growth cone pathfinding and target recognition,followed by latter refinement and remodeling of such projections byevents that require neuronal activity, Goodman and Shatz, Cell/Neuron[Suppl.] 72(10):77–98 (1993). It is further evident that differentneurons extend nerve fibers that are biochemically distinct and rely onspecific guidance cues provided by cell-cell, cell-matrix, andchemotrophic interactions to reach their appropriate synaptic targets,Goodman et al., supra.

One particular means by which diversity of the neuronal cell surface maybe generated is through differential expression of cell surface proteinsreferred to as cell adhesion molecules (CAMs). Neuronally expressed CAMshave been implicated in diverse developmental processes, includingmigration of neurons along radial glial cells, providing permissive orrepulsive substrates for neurite extension, and in promoting theselective fasciculation of axons in projectional pathways. Jessel,Neuron 1: 3–13 (1988); Edelman and Crossin, Annu. Rev. Biochem. 60:155–190 (1991). Interactions between CAMs present on the growth conemembrane and molecules on opposing cell membranes or in theextracellular matrix are thought to provide the specific guidance cuesthat direct nerve fiber outgrowth along appropriate projectionalpathways. Such interactions are likely to result in the activation ofvarious second messenger systems within the growth cone that regulateneurite outgrowth. Doherty and Walsh, Curr. Opin Neurobiol. 2: 595–601(1992).

In higher vertebrates, most neural CAMs have been found to be members ofthree major structural families of proteins: the integrins, thecadherins, and the immunoglobulin gene superfamily (IgSF). Jessel,supra.; Takeichi, Annu. Rev. Biochem. 59: 237–252 (1990); Reichardt andTomaselli, Annu. Rev. Neurosci. 14: 531–570 (1991). Cell adhesionmolecules of the IgSF (or Ig-CAMs), in particular, constitute a largefamily of proteins frequently implicated in neural cell interactions andnerve fiber outgrowth during development, Salzer and Colman, Dev.Neurosci. 11: 377–390 (1989); Brümmendorf and Rathjen, J. Neurochem. 61:1207–1219 (1993). However, the majority of mammalian Ig-CAMs appear tobe too widely expressed to specify navigational pathways or synaptictargets suggesting that other CAMs, yet to be identified, have role inthese more selective interactions of neurons.

Many of the known neural Ig-CAMs have been found to be attached to theplasma membrane via a glycosylphosphatidylinositol (GPI) anchor.Additionally, many studies have implicated GPI-anchored proteins inproviding specific guidance cues during the outgrowth on neurons inspecific pathways. In studies of the grasshopper nervous system,treatment of embryos with phosphatidylinositol-specific phopholipase C(PIPLC), which selectively removes GPI-anchored proteins from thesurfaces of cells, resulted in misdirection and faulty navigation amongsubsets of pioneering growth cones, as well as inhibited migratorypatterns of a subset of early neurons, Chang et al., Devel. 114: 507–519(1992). The projection of retinal fibers to the optic tectum appears todepend, in part, on a 33 kDa GPI-anchored protein, however, the precisenature of this protein is unknown. Stahl et al., Neuron 5: 735–743(1990).

The expression of various GPI-anchored proteins has been characterizedamongst the different populations of primary rat neurons amongst dorsalroot ganglion, sympathetic neurons of the cervical ganglion, sympatheticneurons of the superior cervical ganglion, and cerebellar granuleneurons. Rosen et al., J. Cell Biol. 117: 617–627 (1992). In contrast tothe similar pattern of total membrane protein expression by thesedifferent types of neurons, striking differences were observed in theexpression of GPI-anchored proteins between these neurons. Recently, a65 kDa protein band known as neurotrimin was discovered and found to bedifferentially expressed by primary neurons (Rosen et al., supra), andrestricted to the nervous system and found to be the most abundant andearliest expressed of the GPI-anchored species in the CNS. Struyk etal., J. Neuroscience 15(3):2141–2156 (1995). The discovery ofneurotrimin has further lead to the identification of a family of IgSFmembers, each containing three Ig-like domains that share significantamino acid identity, now termed IgLON. Struyk et al., supra; Pimenta etal., Gene 170(2):189–95 (1996).

Additional members of the IgLON subfamily include opiate binding celladhesion molecule (OBCAM), Schofield et al., EMBO J. 8: 489–495 (1989);limbic associated membrane protein (LAMP), Pimenta et al., supra;CEPU-1; GP55, Wilson et al., J. Cell Sci. 109: 3129–3138 (1996); Eur. J.Neurosci. 9(2):334–41 (1997); and AvGp50, Hancox et al., Brain Res. Mol.Brain Res. 44(2):273–85 (1997).

While the expression of neurotrimin appears to be widespread, it doesappear to correlated with the development of several neural circuits.For example, between E18 and P10, neurotimin mRNA expression within theforebrain is maintained at high levels in neurons of the developingthalamus, cortical subplate, and cortex, particularly laminae V and VI(with less intense expression in II, II, and IV, and minimal expressionin lamina I). Cortical subplate neurons may provide an early, temporaryscaffold for the ingrowing thalamic afferents en route to their finalsynaptic targets in the cortex. Allendoerfer and Shatz, Annu. Rev.Neurosci. 17: 185–218 (1994). Conversely, subplate neurons have beensuggested to be required for cortical neurons from layer V to select VIto grow into the thalamus, and neurons from layer V to select theirtargets in the colliculus, pons, and spinal cord (McConnell et al., J.Neurosci. 14: 1892–1907 (1994). The high level expression of neurotriminin many of these projections suggests that it could be involved in theirdevelopment.

In the hindbrain, high levels of neurotrimin message expression wereobserved within the pontine nucleus and by the internal granule cellsand Purkinje cells of the cerebellum. The pontine nucleus receivedafferent input from a variety of sources including corticopontine fibersof layer V, and is a major source of afferent input, via mossy fibers,to the granule cells which, in turn, are a major source of afferentinput via parallel fibers to Purkinje cells. [Palay and Chan-Palay, Thecerebellar cortex: cytology and organization. New York: Springer (1974].High level expression of neurotrimin these neurons again suggestspotential involvement in the establishment of these circuits.

Neurotrimin also exhibits a graded expression pattern in the earlypostnatal striatum. Increased neurotrimin expression is found overlyingthe dorsolateral striatum of the rat, while lesser hybridizationintensity is seen overlying the ventromedial striatum. Struyk et al.,supra. This region of higher neurotrimin hybridization intensity doesnot correspond to a cytoarchitecturally differentiable region, rather itcorresponds to the primary area of afferent input from layer VI of thecontralateral sensorimotor cortex (Gerfen, Nature 311: 461–464 (1984);Donoghue and Herkenham, Brain Res. 365: 397–403 (1986)). Theventromedial striatum, by contrast, receives the majority of itsafferent input from the perirhinal and association cortex. It isnoteworthy that a complementary graded pattern of LAMP expression, hasbeen observed within the striatium, with highest expression inventromedial regions, and lowest expression dorsolaterally. Levitt,Science 223: 299–301 (1985); Chesselet et al., Neuroscience 40: 725–733(1991).

87. PRO403

Type II transmembrane proteins, also known as single pass transmembraneproteins have an N-terminal portion lodged in the cytoplasm while theC-terminal portion is exposed to the extracellular domain.

Endothelin is a family of vasoconstrictor peptides about which muchactivity has been focused to better understand its basicpharmacological, biochemical and molecular biological features,including the presence and structure of isopeptides and their genes(endothelin-1, -2 and û3), regulation of gene expression, intracellularprocessing, specific endothelin converting enzymes (ECE), receptorsubtypes (ET-A and ET-B), intracellular signal transduction followingreceptor activation, etc.

The endothelin (ET) family of peptides have potent vascular, cardiac andrenal actions which may be of pathophysiological importance in manyhuman disease states. ET-1 is expressed as an inactive 212 amino acidprepropeptide. The prepropeptide is first cleaved at Arg52-Cys53 andArg92-Ala93 and then the carboxy terminal Lys91 and Arg92 are trimmedfrom the protein to generate the propeptide big ET-1.

Endothelin is generated from inactive intermediates, the bigendothelins, by a unique processing event catalyzed by the zincmetalloprotease, endothelin converting enzyme (ECE). ECE was recentlycloned, and its structure was shown to be a single pass transmembraneprotein with a short intracellular N-terminal and a long extracellularC-terminal that contains the catalytic domain and numerousN-glycosylation sites. ECEs cleave the endothelin propeptide betweenTrp73 and Val74 producing the active peptide, ET, which appears tofunction as a local rather than a circulating hormone (Rubanyi, G. M. &Polokoff, M. A., Pharmachological Reviews 46: 325–415 (1994). Thus ECEactivity is a potential site of regulation of endothelin production anda possible target for therapeutic intervention in the endothelin system.By blocking ECE activity, it is possible stop the production of ET-1 byinhibiting the conversion of the relatively inactive precursor, bigET-1, to the physiologically active form.

Endothelins may play roles in the pathophysiology of a number of diseasestates including: 1) cardiovascular diseases (vasospasm, hypertension,myocardial ischemia; reperfusion injury and acute myochardialinfarction, stroke (cerebral ischemia), congestive heart failure, shock,atherosclerosis, vascular thickening); 2) kidney disease (acute andchronic renal failure, glomerulonephritis, cirrhosis); 3) lung disease(bronchial asthma, pulmonary hypertension); 4) gastrointestinaldisorders (gastric ulcer, inflammatory bowel diseases); 5) reproductivedisorders (premature labor, dysmenorhea, preeclampsia) and 6)carcinogenesis. Rubanyi & Polokoff, supra.

SUMMARY OF THE INVENTION

1. PRO213

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO213”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO213 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO213 polypeptidehaving amino acid residues 1 to 295 of FIG. 2 (SEQ ID NO:2), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO213polypeptide. In particular, the invention provides isolated nativesequence PRO213 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 295 of FIG. 2 (SEQ ID NO:2).

2. PRO274

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO274”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO274 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO274 polypeptidehaving amino acid residues 1 to 492 of FIG. 4 (SEQ ID NO:7), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA39987-1184 vector deposited on Apr. 21, 1998as ATCC 209786 which includes the nucleotide sequence encoding PRO274.

In another embodiment, the invention provides isolated PRO274polypeptide. In particular, the invention provides isolated nativesequence PRO274 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 492 of FIG. 4 (SEQ ID NO:7). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO274 polypeptide. Optionally, thePRO274 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA39987-1184 vectordeposited on Apr. 21, 1998 as ATCC 209786.

In another embodiment, the invention provides three expressed sequencetags (EST) comprising the nucleotide sequences of SEQ ID NO:8 (hereindesignated as DNA17873), SEQ ID NO:9 (herein designated as DNA36157) andSEQ ID NO:10 (herein designated as DNA28929) (see FIG. 5-7,respectively).

3. PRO300

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO300”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO300 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO300 polypeptidehaving amino acid residues 1 to 457 of FIG. 9 (SEQ ID NO:19), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA40625-1189 vector deposited on Apr. 21, 1998as ATCC 209788 which includes the nucleotide sequence encoding PRO300.

In another embodiment, the invention provides isolated PRO300polypeptide. In particular, the invention provides isolated nativesequence PRO300 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 457 of FIG. 9 (SEQ ID NO:19). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO300 polypeptide. Optionally, thePRO300 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA40625-1189 vectordeposited on Apr. 21, 1998 as ATCC 209788.

4. PRO284

Applicants have identified a cDNA clone that encodes a noveltransmembrane polypeptide, wherein the polypeptide is designated in thepresent application as “PRO284”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO284 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO284 polypeptidehaving amino acid residues 1 to 285 of FIG. 11 (SEQ ID NO:28), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO284 polypeptide having amino acid residuesabout 25 to 285 of FIG. 11 (SEQ ID NO:28) or 1 or about 25 to X of FIG.11 (SEQ ID NO:28), where X is any amino acid from 71 to 80 of FIG. 11(SEQ ID NO:28), or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions. The isolated nucleic acidsequence may comprise the cDNA insert of the DNA23318-1211 vectordeposited on Apr. 21, 1998 as ATCC 209787 which includes the nucleotidesequence encoding PRO284.

In another embodiment, the invention provides isolated PRO284polypeptide. In particular, the invention provides isolated nativesequence PRO284 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 285 of FIG. 11 (SEQ ID NO:28).Additional embodiments of the present invention are directed to isolatedPRO284 polypeptides comprising amino acids about 25 to 285 of FIG. 11(SEQ ID NO:28) or 1 or about 25 to X of FIG. 11 (SEQ ID NO:28), where Xis any amino acid from 71 to 80 of FIG. 11 (SEQ ID NO:28). Optionally,the PRO284 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA23318-1211 vectordeposited on Apr. 21, 1998 as ATCC 209787.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA12982 which comprises the nucleotidesequence of SEQ ID NO:29.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA15886 which comprises the nucleotidesequence of SEQ ID NO:30.

5. PRO296

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the sarcoma-amplified protein SAS, wherein thepolypeptide is designated in the present application as “PRO296”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO296 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO296 polypeptidehaving amino acid residues 1 to 204 of FIG. 15 (SEQ ID NO:36), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO296 polypeptide having amino acid residuesabout 35 to 204 of FIG. 15 (SEQ ID NO:36) or amino acid 1 or about 35 toX of FIG. 15 (SEQ ID NO:36), where X is any amino acid from 42 to 51 ofFIG. 15 (SEQ ID NO:36), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. The isolated nucleicacid sequence may comprise the cDNA insert of the DNA39979-1213 vectordeposited on Apr. 21, 1998 as ATCC 209789 which includes the nucleotidesequence encoding PRO296.

In another embodiment, the invention provides isolated PRO296polypeptide. In particular, the invention provides isolated nativesequence PRO296 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 204 of FIG. 15 (SEQ ID NO:36).Additional embodiments of the present invention are directed to PRO296polypeptides comprising amino acids about 35 to 204 of FIG. 15 (SEQ IDNO:36) or amino acid 1 or about 35 to X of FIG. 15 (SEQ ID NO:36), whereX is any amino acid from 42 to 51 of FIG. 15 (SEQ ID NO:36). Optionally,the PRO296 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA39979-1213 vectordeposited on Apr. 21, 1998 as ATCC 209789.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA23020 comprising the nucleotide sequenceof SEQ ID NO:37.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA21971 comprising the nucleotide sequenceof SEQ ID NO:38.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA29037 comprising the nucleotide sequenceof SEQ ID NO:39.

6. PRO329

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to a high affinity immunoglobulin F_(c) receptor,wherein the polypeptide is designated in the present application as“PRO329”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO329 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO329 polypeptidehaving amino acid residues 1 to 359 of FIG. 20 (SEQ ID NO:45), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA40594-1233 vector deposited on Feb. 5, 1998 asATCC 209617 which includes the nucleotide sequence encoding PRO329.

In another embodiment, the invention provides isolated PRO329polypeptide. In particular, the invention provides isolated nativesequence PRO329 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 359 of FIG. 20 (SEQ ID NO:45).Optionally, the PRO329 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA40594-1233 vector deposited on Feb. 5, 1998 as ATCC 209617.

7. PRO362

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to A33 antigen and HCAR membrane-bound protein, whereinthe polypeptide is designated in the present application as “PRO362”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO362 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO362 polypeptidehaving amino acid residues 1 to 321 of FIG. 22 (SEQ ID NO:52), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO362 polypeptide having amino acid residues1 to X of FIG. 22 (SEQ ID NO:52) where X is any amino acid from aminoacid 271 to 280, or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions. The isolated nucleic acidsequence may comprise the cDNA insert of the DNA45416-1251 vectordeposited on Feb. 5, 1998 as ATCC 209620 which includes the nucleotidesequence encoding PRO362.

In another embodiment, the invention provides isolated PRO362polypeptide. In particular, the invention provides isolated nativesequence PRO362 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 321 of FIG. 22 (SEQ ID NO:52). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO362 polypeptide comprising aminoacids 1 to X of the amino acid sequence shown in FIG. 22 (SEQ ID NO:52),wherein X is any amino acid from amino acid 271 to 280. Optionally, thePRO362 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA45416-1251 vectordeposited on Feb. 5, 1998 as ATCC 209620.

8. PRO363

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the cell surface receptor protein HCAR, wherein thepolypeptide is designated in the present application as “PRO363”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO363 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO363 polypeptidehaving amino acid residues 1 to 373 of FIG. 24 (SEQ ID NO:59), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding a PRO363 extracellular domain polypeptide havingamino acid residues 1 to X of FIG. 24 (SEQ ID NO:59) where X is anyamino acid from amino acid 216 to amino acid 225, or is complementary tosuch encoding nucleic acid sequence, and remains stably bound to itunder at least moderate, and optionally, under high stringencyconditions. The isolated nucleic acid sequence may comprise the cDNAinsert of the DNA45419-1252 vector deposited on Feb. 5, 1998 as ATCC209616 which includes the nucleotide sequence encoding PRO363.

In another embodiment, the invention provides isolated PRO363polypeptide. In particular, the invention provides isolated nativesequence PRO363 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 373 of FIG. 24 (SEQ ID NO:59). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO363 polypeptide, wherein thatextracellular domain may comprise amino acids 1 to X of the sequenceshown in FIG. 24 (SEQ ID NO:59), where X is any amino acid from aminoacid 216 to 225. Optionally, the PRO363 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA45419-1252 vector deposited on Feb. 5, 1998 as ATCC 209616.

9. PRO868

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to tumor necrosis factor receptor, wherein thepolypeptide is designated in the present application as “PRO868”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO868 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO868 polypeptidehaving amino acid residues 1 to 655 of FIG. 26 (SEQ ID NO:64), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO868 polypeptide having amino acid residues1 to X of FIG. 26 (SEQ ID NO:64), where X is any amino acid from aminoacid 343 to 352 of the sequence shown in FIG. 26 (SEQ ID NO:64), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In yet another aspect, the isolated nucleic acidcomprises DNA encoding the PRO868 polypeptide having amino acid residuesX to 655 of FIG. 26 (SEQ ID NO:64), where X is any amino acid from aminoacid 371 to 380 of the sequence shown in FIG. 26 (SEQ ID NO:64), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA52594-1270 vector deposited on Mar. 17, 1998as ATCC 209679 which includes the nucleotide sequence encoding PRO868.

In another embodiment, the invention provides isolated PRO868polypeptide. In particular, the invention provides isolated nativesequence PRO868 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 655 of FIG. 26 (SEQ ID NO:64). Inanother aspect, the isolated PRO868 polypeptide comprises amino acidresidues 1 to X of FIG. 26 (SEQ ID NO:64), where X is any amino acidfrom amino acid 343 to 352 of the sequence shown in FIG. 26 (SEQ IDNO:64). In yet another aspect, the PRO868 polypeptide comprises aminoacid residues X to 655 of FIG. 26 (SEQ ID NO:64), where X is any aminoacid from amino acid 371 to 380 of the sequence shown in FIG. 26 (SEQ IDNO:64). Optionally, the PRO868 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA52594-1270 vector deposited on Mar. 17, 1998 as ATCC 209679.

10. PRO382

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to serine proteases, wherein the polypeptide isdesignated in the present application as “PRO382”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO382 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO382 polypeptidehaving amino acid residues 1 to 453 of FIG. 28 (SEQ ID NO:69), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA45234-1277 vector deposited on Mar. 5, 1998 asATCC 209654 which includes the nucleotide sequence encoding PRO382.

In another embodiment, the invention provides isolated PRO382polypeptide. In particular, the invention provides isolated nativesequence PRO382 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 453 of FIG. 28 (SEQ ID NO:69). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO382 polypeptide, with or withoutthe signal peptide. Optionally, the PRO382 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA45234-1277 vector deposited on Mar. 5, 1998 as ATCC 209654.

11. PRO545

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to meltrin, wherein the polypeptide is designated in thepresent application as “PRO545”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO545 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO545 polypeptidehaving amino acid residues 1 to 735 of FIG. 30 (SEQ ID NO:74), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Mar. 5, 1998 as ATCC 209655which includes the nucleotide sequence encoding PRO545.

In another embodiment, the invention provides isolated PRO545polypeptide. In particular, the invention provides isolated nativesequence PRO545 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 735 of FIG. 30 (SEQ ID NO:74). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO545 polypeptide. Optionally, thePRO545 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Mar.5, 1998 as ATCC 209655.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA13217 comprising the nucleotide sequenceof SEQ ID NO:75 (FIG. 31).

12. PRO617

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to CD24, wherein the polypeptide is designated in thepresent application as “PRO617”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO617 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO617 polypeptidehaving amino acid residues 1 to 67 of FIG. 33 (SEQ ID NO:85), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA48309-1280 vector deposited on Mar. 5, 1998 asATCC 209656 which includes the nucleotide sequence encoding PRO617.

In another embodiment, the invention provides isolated PRO617polypeptide. In particular, the invention provides isolated nativesequence PRO617 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 67 of FIG. 33 (SEQ ID NO:85).Optionally, the PRO617 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA48309-1280 vector deposited on Mar. 5, 1998 as ATCC 209656.

13. PRO700

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to protein disulfide isomerase, wherein thepolypeptide is designated in the present application as “PRO700”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO700 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO700 polypeptidehaving amino acid residues 1 to 432 of FIG. 35 (SEQ ID NO:90), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO700 polypeptide having amino acid residuesfrom about 34 to 432 of FIG. 35 (SEQ ID NO:90), or is complementary tosuch encoding nucleic acid sequence, and remains stably bound to itunder at least moderate, and optionally, under high stringencyconditions. The isolated nucleic acid sequence may comprise the cDNAinsert of the vector deposited on Mar. 31, 1998 as ATCC 209721 whichincludes the nucleotide sequence encoding PRO700.

In another embodiment, the invention provides isolated PRO700polypeptide. In particular, the invention provides isolated nativesequence PRO700 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 432 of FIG. 35 (SEQ ID NO:90). Inanother embodiment, the invention provides an isolated PRO700polypeptide absent the signal sequence, which includes an amino acidsequence comprising residues from about 34 to 432 of FIG. 35 (SEQ IDNO:90). Optionally, the PRO700 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of the vectordeposited on Mar. 31, 1998 as ATCC 209721.

14. PRO702

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to conglutinin, wherein the polypeptide is designated inthe present application as “PRO702”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO702 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO702 polypeptidehaving amino acid residues 1 to 277 of FIG. 37 (SEQ ID NO:97), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO702 polypeptide having amino acid residues26 to 277 of FIG. 37 (SEQ ID NO:97), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA50980-1286 vector deposited on Mar. 31, 1998 as ATCC 209717 whichincludes the nucleotide sequence encoding PRO702.

In another embodiment, the invention provides isolated PRO702polypeptide. In particular, the invention provides isolated nativesequence PRO702 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 277 of FIG. 37 (SEQ ID NO:97). Anadditional embodiment of the present invention is directed to anisolated PRO702 polypeptide comprising amino acid residues 26 to 277 ofFIG. 37 (SEQ ID NO:97). Optionally, the PRO702 polypeptide is obtainedor is obtainable by expressing the polypeptide encoded by the cDNAinsert of the DNA50980-1286 vector deposited on Mar. 31, 1998 as ATCC209717.

15. PRO703

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to VLCAS, wherein the polypeptide isdesignated in the present application as “PRO703”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO703 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO703 polypeptidehaving amino acid residues 1 to 730 of FIG. 39 (SEQ ID NO:102), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO703 polypeptide having amino acid residuesfrom about 43 to 730 of FIG. 39 (SEQ ID NO:102), or is complementary tosuch encoding nucleic acid sequence, and remains stably bound to itunder at least moderate, and optionally, under high stringencyconditions. The isolated nucleic acid sequence may comprise the cDNAinsert of the DNA50913-1287 vector deposited on Mar. 31, 1998 as ATCC209716 which includes the nucleotide sequence encoding PRO703.

In another embodiment, the invention provides isolated PRO703polypeptide. In particular, the invention provides isolated nativesequence PRO703 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 730 of FIG. 39 (SEQ ID NO:102).In another embodiment, the invention provides an isolated PRO703polypeptide absent the signal sequence, which includes an amino acidsequence comprising residues from about 43 to 730 of FIG. 30 (SEQ IDNO:102). Optionally, the PRO730 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA50913-1287 vector deposited on Mar. 31, 1998 as ATCC 209716.

16. PRO705

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to K-glypican, wherein the polypeptide is designated inthe present application as “PRO705”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO705 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO705 polypeptidehaving amino acid residues 1 to 555 of FIG. 41 (SEQ ID NO:109), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO705 polypeptide having amino acid residuesabout 24 to 555 of FIG. 41 (SEQ ID NO:109), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA50914-1289 vector deposited on Mar. 31, 1998 as ATCC 209722 whichincludes the nucleotide sequence encoding PRO705.

In another embodiment, the invention provides isolated PRO705polypeptide. In particular, the invention provides isolated nativesequence PRO705 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 555 of FIG. 41 (SEQ ID NO:109).An additional embodiment of the present invention is directed to anisolated PRO705 polypeptide comprising amino acid residues about 24 to555 of FIG. 41 (SEQ ID NO:109). Optionally, the PRO705 polypeptide isobtained or is obtainable by expressing the polypeptide encoded by thecDNA insert of the DNA50914-1289 vector deposited on Mar. 31, 1998 asATCC 209722.

17. PRO708

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the aryl sulfatases, wherein the polypeptide isdesignated in the present application as “PRO708”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO708 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO708 polypeptidehaving amino acid residues 1 to 515 of FIG. 43 (SEQ ID NO:114), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA48296-1292 vector deposited on Mar. 11, 1998as ATCC 209668 which includes the nucleotide sequence encoding PRO708.

In another embodiment, the invention provides isolated PRO708polypeptide. In particular, the invention provides isolated nativesequence PRO708 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 515 of FIG. 43 (SEQ ID NO:114).Another embodiment is directed to a PRO708 polypeptide comprisingresidues 38–515 of the amino acid sequence shown in FIG. 43 (SEQ IDNO:114). Optionally, the PRO708 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA48296-1292 vector deposited on Mar. 11, 1998 as ATCC 209668.

18. PRO320

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to fibulin, wherein the polypeptide is designated in thepresent application as “PRO320”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO320 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO320 polypeptidehaving amino acid residues 1 to 338 of FIG. 45 (SEQ ID NO:119), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Mar. 11, 1998 as ATCC 209670which includes the nucleotide sequence encoding PRO320.

In another embodiment, the invention provides isolated PRO320polypeptide. In particular, the invention provides isolated nativesequence PRO320 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 338 of FIG. 45 (SEQ ID NO:119).Optionally, the PRO320 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of the vectordeposited on Mar. 11, 1998 as ATCC 209670.

19. PRO324

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to oxidoreductases, wherein the polypeptide isdesignated in the present application as “PRO324”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO324 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO324 polypeptidehaving amino acid residues 1 to 289 of FIG. 47 (SEQ ID NO:124), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO324 polypeptide having amino acid residues1 or about 32 to X of FIG. 47 (SEQ ID NO:124), where X is any amino acidfrom 131 to 140, or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions. The isolated nucleic acidsequence may comprise the cDNA insert of the DNA36343-1310 vectordeposited on Mar. 30, 1998 as ATCC 209718 which includes the nucleotidesequence encoding PRO324.

In another embodiment, the invention provides isolated PRO324polypeptide. In particular, the invention provides isolated nativesequence PRO324 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 289 of FIG. 47 (SEQ ID NO:124).The invention also provides isolated PRO324 polypeptide comprisingresidues 1 or about 32 to X of FIG. 47 (SEQ ID NO:124), wherein X is anyamino acid from about 131–140. Optionally, the PRO324 polypeptide isobtained or is obtainable by expressing the polypeptide encoded by thecDNA insert of the DNA36343-1310 vector deposited on Mar. 30, 1998 asATCC 209718.

20. PRO351

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to prostasin, wherein the polypeptide isdesignated in the present application as “PRO351”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO351 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO351 polypeptidehaving amino acid residues 1 to 571 of FIG. 49 (SEQ ID NO:132), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO351 polypeptide having amino acid residuesabout 16 to 571 of FIG. 49 (SEQ ID NO:132), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA40571-1315 vector deposited on Apr. 21, 1998 as ATCC 209784 whichincludes the nucleotide sequence encoding PRO351.

In another embodiment, the invention provides isolated PRO351polypeptide. In particular, the invention provides isolated nativesequence PRO351 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 571 of FIG. 49 (SEQ ID NO:132).In another embodiment, the invention provides an isolated PRO351polypeptide absent the signal sequence, which includes an amino acidsequence comprising residues from about 16 to 571 of FIG. 49 (SEQ IDNO:132). Optionally, the PRO351 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA40571-1315 vector deposited on Apr. 21, 1998 as ATCC 209784.

21. PRO352

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to butyrophilin, wherein the polypeptide is designatedin the present application as “PRO352”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO352 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO352 polypeptidehaving amino acid residues 1 to 316 of FIG. 51 (SEQ ID NO:137), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO352 polypeptide having amino acid residuesof about 29 to 316 of FIG. 51 (SEQ ID NO:137), or 1 or about 29 to X ofFIG. 51, where X is any amino acid from 246 to 255, or is complementaryto such encoding nucleic acid sequence, and remains stably bound to itunder at least moderate, and optionally, under high stringencyconditions. The isolated nucleic acid sequence may comprise the cDNAinsert of the DNA41386-1316 vector deposited on Mar. 26, 1998 as ATCC209703 which includes the nucleotide sequence encoding PRO352.

In another embodiment, the invention provides isolated PRO352polypeptide. In particular, the invention provides isolated nativesequence PRO352 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 316 of FIG. 51 (SEQ ID NO:137).In other embodiments, the invention provides isolated PRO352 polypeptidecomprising residues about 29 to 316 of FIG. 51 (SEQ ID NO:137) and 1 orabout 29 to X of FIG. 51 (SEQ ID NO:137), wherein X is any amino acidfrom 246 to 255. Optionally, the PRO352 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA41386-1316 vector deposited on Mar. 26, 1998 as ATCC 209703.

22. PRO381

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to immunophilin proteins, wherein the polypeptide isdesignated in the present application as “PRO381”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO381 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO381 polypeptidehaving amino acid residues 1 to 211 of FIG. 53 (SEQ ID NO:145), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO381 polypeptide having amino acid residuesabout 21 to 211 of FIG. 53 (SEQ ID NO:145), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA44194-1317 vector deposited on Apr. 28, 1998 as ATCC 209808 whichincludes the nucleotide sequence encoding PRO381.

In another embodiment, the invention provides isolated PRO381polypeptide. In particular, the invention provides isolated nativesequence PRO381 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 211 of FIG. 53 (SEQ ID NO:145).Another embodiment is directed to a PRO381 polypeptide comprising aminoacids about 21 to 211 of FIG. 53 (SEQ ID NO:145). Optionally, the PRO381polypeptide is obtained or is obtainable by expressing the polypeptideencoded by the cDNA insert of the DNA44194-1317 vector deposited on Apr.28, 1998 as ATCC 209808.

23. PRO386

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the beta-2 subunit of a sodium channel, wherein thepolypeptide is designated in the present application as “PRO386”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO386 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO386 polypeptidehaving amino acid residues 1 to 215 of FIG. 55 (SEQ ID NO:150), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO386 polypeptide having amino acid residuesabout 21 to 215 of FIG. 55 (SEQ ID NO:150) or 1 or about 21 to X, whereX is any amino acid from 156 to 165 of FIG. 55 (SEQ ID NO:150), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA45415-1318 vector deposited on Apr. 28, 1998as ATCC 209810 which includes the nucleotide sequence encoding PRO386.

In another embodiment, the invention provides isolated PRO386polypeptide. In particular, the invention provides isolated nativesequence PRO386 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 215 of FIG. 55 (SEQ ID NO:150).Other embodiments of the present invention are directed to PRO386polypeptides comprising amino acids about 21 to 215 of FIG. 55 (SEQ IDNO:150) and 1 or about 21 to X of FIG. 55 (SEQ ID NO:150), wherein X isany amino acid from 156 to 165 of FIG. 55 (SEQ ID NO:150). Optionally,the PRO386 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA45415-1318 vectordeposited on Apr. 28, 1998 as ATCC 209810.

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of SEQ ID NO:151 whichcorresponds to an EST designated herein as DNA23350.

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of SEQ ID NO:152 whichcorrsponds to an EST designated herein as DNA23536.

24. PRO540

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to LCAT, wherein the polypeptide isdesignated in the present application as “PRO540”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO540 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO540 polypeptidehaving amino acid residues 1 to 412 of FIG. 59 (SEQ ID NO:157), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO540 polypeptide having amino acid residuesabout 29 to 412 of FIG. 59 (SEQ ID NO:157), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA44189-1322 vector deposited on Mar. 26, 1998 as ATCC 209699 whichincludes the nucleotide sequence encoding PRO540.

In another embodiment, the invention provides isolated PRO540polypeptide. In particular, the invention provides isolated nativesequence PRO540 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 412 of FIG. 59 (SEQ ID NO:157).The invention also provides isolated PRO540 polypeptide, which in oneembodiment, includes an amino acid sequence comprising residues about 29to 412 of FIG. 59 (SEQ ID NO:157). Optionally, the PRO540 polypeptide isobtained or is obtainable by expressing the polypeptide encoded by thecDNA insert of the DNA44189-1322 vector deposited on Mar. 26, 1998 asATCC 209699.

25. PRO615

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to synaptogyrin, wherein the polypeptide isdesignated in the present application as “PRO615”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO615 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO615 polypeptidehaving amino acid residues 1 to 224 of FIG. 61 (SEQ ID NO:162), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO615 polypeptide having amino acid residuesX to 224 of FIG. 61 (SEQ ID NO:162), where X is any amino acid from 157to 166, or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. The isolated nucleic acid sequence maycomprise the cDNA insert of the DNA48304-1323 vector deposited on Apr.28, 1998 as ATCC 209811 which includes the nucleotide sequence encodingPRO615.

In another embodiment, the invention provides isolated PRO615polypeptide. In particular, the invention provides isolated nativesequence PRO615 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 224 of FIG. 61 (SEQ ID NO:162).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO615 polypeptide which comprisesamino acid residues X to 224 of FIG. 61 (SEQ ID NO:162), where X is anyamino acid from 157 to 166 of FIG. 61 (SEQ ID NO:162). Optionally, thePRO615 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA48304-1323 vectordeposited on Apr. 28, 1998 as ATCC 209811.

26. PRO618

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to enteropeptidase, wherein the polypeptideis designated in the present application as “PRO618”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO618 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO618 polypeptidehaving amino acid residues 1 to 802 of FIG. 63 (SEQ ID NO:169), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding an isolated extracellular domain of a PRO618polypeptide having amino acid residues X to 802 of FIG. 63 (SEQ IDNO:169), where X is any amino acid from 63 to 72 of FIG. 63 (SEQ IDNO:169), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. The isolated nucleic acid sequence maycomprise the cDNA insert of the DNA49152-1324 vector deposited on Apr.28, 1998 as ATCC 209813 which includes the nucleotide sequence encodingPRO618.

In another embodiment, the invention provides isolated PRO618polypeptide. In particular, the invention provides isolated nativesequence PRO618 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 802 of FIG. 63 (SEQ ID NO:169).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO618 polypeptide comprising aminoacid X to 802 where X is any amino acid from 63 to 72 of FIG. 63 (SEQ IDNO:169). Optionally, the PRO618 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA49152-1324 vector deposited on Apr. 28, 1998 as ATCC 209813.

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of SEQ ID NO:170, designatedherein as DNA35597 (see FIG. 64).

27. PRO719

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to lipoprotein lipase H, wherein the polypeptide isdesignated in the present application as “PRO719”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO719 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO719 polypeptidehaving amino acid residues 1 to 354 of FIG. 66 (SEQ ID NO:178), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO719 polypeptide having amino acid residuesabout 17 to 354 of FIG. 66 (SEQ ID NO:178), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA49646-1327 vector deposited on Mar. 26, 1998 as ATCC 209705 whichincludes the nucleotide sequence encoding PRO719.

In another embodiment, the invention provides isolated PRO719polypeptide. In particular, the invention provides isolated nativesequence PRO719 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 354 of FIG. 66 (SEQ ID NO:178).In another embodiment, the invention provides isolated PRO719polypeptide which comprises residues about 17 to 354 of FIG. 66 (SEQ IDNO:178). Optionally, the PRO719 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA49646-1327 vector deposited on Mar. 26, 1998 as ATCC 209705.

28. PRO724

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the LDL receptor, wherein the polypeptide isdesignated in the present application as “PRO724”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO724 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO724 polypeptidehaving amino acid residues 1 to 713 of FIG. 68 (SEQ ID NO:183), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding a soluble PRO724 polypeptide having amino acidresidues 1 to X of FIG. 68 (SEQ ID NO:183) where X is any amino acidfrom amino acid 437 to 446, or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. The above twopolypeptides may either possess or not possess the signal peptide. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA49631-1328 vector deposited on Apr. 28, 1998 as ATCC 209806 whichincludes the nucleotide sequence encoding PRO724.

In another embodiment, the invention provides isolated PRO724polypeptide. In particular, the invention provides isolated nativesequence PRO724 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 713 of FIG. 68 (SEQ ID NO:183).In another embodiment, the invention provides isolated soluble PRO724polypeptide. In particular, the invention provides isolated solublePRO724 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 to X of FIG. 68 (SEQ ID NO:183), where Xis any amino acid from 437 to 446 of the sequence shown in FIG. 68 (SEQID NO:183). Optionally, the PRO724 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA49631-1328 vector deposited on Apr. 28, 1998 as ATCC 209806.

29. PRO772

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to A4 protein, wherein the polypeptide is designated inthe present application as “PRO772”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO772 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO772 polypeptidehaving amino acid residues 1 to 152 of FIG. 70 (SEQ ID NO:190), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO772 polypeptide having amino acid residues1 to X of FIG. 70 (SEQ ID NO:190), where X is any amino acid from 21 to30 of FIG. 70 (SEQ ID NO:190), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA49645-1347vector deposited on Apr. 28, 1998 as ATCC 209809 which includes thenucleotide sequence encoding PRO772.

In another embodiment, the invention provides isolated PRO772polypeptide. In particular, the invention provides isolated nativesequence PRO772 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 152 of FIG. 70 (SEQ ID NO:190).Additional embodiments of the present invention are directed to PRO772polypeptides comprising amino acids 1 to X of FIG. 70 (SEQ ID NO:190),where X is any amino acid from 21 to 30 of FIG. 70 (SEQ ID NO:190).Optionally, the PRO772 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA49645-1347 vector deposited on Apr. 28, 1998 as ATCC 209809.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA43509 comprising the nucleotide sequenceof SEQ ID NO:191 (FIG. 71).

30. PRO852

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to various protease enzymes, wherein the polypeptide isdesignated in the present application as “PRO852”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO852 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO852 polypeptidehaving amino acid residues 1 to 518 of FIG. 73 (SEQ ID NO:196), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO852 polypeptide having amino acid residuesabout 21 to 518 of FIG. 73 (SEQ ID NO:196) or 1 or about 21 to X of FIG.73 (SEQ ID NO:196) where X is any amino acid from amino acid 461 toamino acid 470 of FIG. 73 (SEQ ID NO:196), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA45493-1349 vector deposited on Apr. 28, 1998 as ATCC 209805 whichincludes the nucleotide sequence encoding PRO852.

In another embodiment, the invention provides isolated PRO852polypeptide. In particular, the invention provides isolated nativesequence PRO852 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 518 of FIG. 73 (SEQ ID NO:196).In other embodiments, the PRO852 comprises amino acids about 21 to aminoacid 518 of FIG. 73 (SEQ ID NO:196) or amino acids 1 or about 21 to X ofFIG. 73 (SEQ ID NO:196), where X is any amino acid from amino acid 461to amino acid 470 of FIG. 73 (SEQ ID NO:196). Optionally, the PRO852polypeptide is obtained or is obtainable by expressing the polypeptideencoded by the cDNA insert of the DNA45493-1349 vector deposited on Apr.28, 1998 as ATCC 209805.

31. PRO853

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to reductase, wherein the polypeptide isdesignated in the present application as “PRO853”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO853 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO853 polypeptidehaving amino acid residues 1 to 377 of FIG. 75 (SEQ ID NO:206), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO853 polypeptide having amino acid residuesabout 17 to 377 of FIG. 75 (SEQ ID NO:206), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA48227-1350 vector deposited on Apr. 28, 1998 as ATCC 209812 whichincludes the nucleotide sequence encoding PRO853.

In another embodiment, the invention provides isolated PRO853polypeptide. In particular, the invention provides isolated nativesequence PRO853 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 377 of FIG. 75 (SEQ ID NO:206).In another embodiment, the invention provides an isolated PRO853polypeptide absent the signal sequence, which includes an amino acidsequence comprising residues from about 17 to 377 of FIG. 75 (SEQ IDNO:206). Optionally, the PRO853 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA48227-1350 vector deposited on Apr. 28, 1998 as ATCC 209812.

32. PRO860

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to neurofascin, wherein the polypeptide isdesignated in the present application as “PRO860”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO860 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO860 polypeptidehaving amino acid residues 1 to 985 of FIG. 77 (SEQ ID NO:211), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO860 polypeptide having amino acid residues1 to X of FIG. 77 (SEQ ID NO:211), where X is any amino acid from443–452 of FIG. 77 (SEQ ID NO:211), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA41404-1352vector deposited on May 6, 1998 as ATCC 209844 which includes thenucleotide sequence encoding PRO860.

In another embodiment, the invention provides isolated PRO860polypeptide. In particular, the invention provides isolated nativesequence PRO860 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 985 of FIG. 77 (SEQ ID NO:211).In another embodiment, the invention provides an isolated PRO860polypeptide which includes an amino acid sequence comprising residues 1to X of FIG. 77 (SEQ ID NO:211), where X is any amino acid residue from443 to 452 of FIG. 77 (SEQ ID NO:211). Optionally, the PRO860polypeptide is obtained or is obtainable by expressing the polypeptideencoded by the cDNA insert of the DNA41404-1352 vector deposited on May6, 1998 as ATCC 209844.

33. PRO846

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to CMRF35, wherein the polypeptide isdesignated in the present application as “PRO846”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO846 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO846 polypeptidehaving amino acid residues 1 to 332 of FIG. 79 (SEQ ID NO:216), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO846 polypeptide having amino acid residuesabout 18 to 332 of FIG. 79 (SEQ ID NO:216) or 1 or about 18 to X of SEQID NO:216, where X is any amino acid from 243 to 252 of FIG. 79 (SEQ IDNO:216), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. The isolated nucleic acid sequence maycomprise the cDNA insert of the DNA44196-1353 vector deposited on May 6,1998 as ATCC 209847 which includes the nucleotide sequence encodingPRO846.

In another embodiment, the invention provides isolated PRO846polypeptide. In particular, the invention provides isolated nativesequence PRO846 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 332 of FIG. 79 (SEQ ID NO:216).In other embodiments, the invention provides an isolated PRO846polypeptide absent the signal sequence, which includes an amino acidsequence comprising residues from about 18 to 332 of FIG. 79 (SEQ IDNO:216). Additional embodiments of the present invention are directed toan isolated PRO846 polypeptide comprising amino acid 1 or about 18 to Xof FIG. 79 (SEQ ID NO:216), where X is any amino acid from 243 to 252 ofFIG. 79 (SEQ ID NO:216). Optionally, the PRO846 polypeptide is obtainedor is obtainable by expressing the polypeptide encoded by the cDNAinsert of the DNA44196-1353 vector deposited on May 6, 1998 as ATCC209847.

34. PRO862

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to lysozyme, wherein the polypeptide isdesignated in the present application as “PRO862”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO862 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO862 polypeptidehaving amino acid residues 1 to 146 of FIG. 81 (SEQ ID NO:221), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO862 polypeptide having amino acid residuesabout 19 to 146 of FIG. 81 (SEQ ID NO:221), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA52187-1354 vector deposited on May 6, 1998 as ATCC 209845 whichincludes the nucleotide sequence encoding PRO862.

In another embodiment, the invention provides isolated PRO862polypeptide. In particular, the invention provides isolated nativesequence PRO862 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 146 of FIG. 81 (SEQ ID NO:221).in another embodiment, the invention provides an isolated PRO862polypeptide absent the signal sequence, which includes an amino acidsequence comprising residues from about 19 to 146 of FIG. 81 (SEQ IDNO:221). Optionally, the PRO862 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA52187-1354 vector deposited on May 6, 1998 as ATCC 209845.

35. PRO864

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence similarity to Wnt-4, wherein the polypeptide isdesignated in the present application as “PRO864”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO864 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO864 polypeptidehaving amino acid residues 1 to 351 of FIG. 83 (SEQ ID NO:226), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO864 polypeptide having amino acid residuesabout 23 to 351 of FIG. 83 (SEQ ID NO:226), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA48328-1355 vector deposited on May 6, 1998 as ATCC 209843 whichincludes the nucleotide sequence encoding PRO864.

In another embodiment, the invention provides isolated PRO864polypeptide. In particular, the invention provides isolated nativesequence PRO864 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 351 of FIG. 83 (SEQ ID NO:226).In another embodiment, the invention provides an isolated PRO864polypeptide absent the signal sequence, which includes an amino acidsequence comprising residues from about 23 to 351 of FIG. 83 (SEQ IDNO:226). Optionally, the PRO864 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA48328-1355 vector deposited on May 6, 1998 as ATCC 209843.

36. PRO792

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to CD23, wherein the polypeptide is designated in thepresent application as “PRO792”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO792 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO792 polypeptidehaving amino acid residues 1 to 293 of FIG. 85 (SEQ ID NO:231), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO792 polypeptide having amino acid residuesX to 293 of FIG. 85 (SEQ ID NO:231) where X is any amino acid from 50 to59 of FIG. 85 (SEQ ID NO:231), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA56352-1358vector deposited on May 6, 1998 as ATCC 209846 which includes thenucleotide sequence encoding PRO792.

In another embodiment, the invention provides isolated PRO792polypeptide. In particular, the invention provides isolated nativesequence PRO792 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 293 of FIG. 85 (SEQ ID NO:231).An additional embodiment of the present invention is directed to PRO792polypeptide comprising amino acids X to 293 of FIG. 85 (SEQ ID NO:231),where X is any amino acid from 50 to 59 of FIG. 85 (SEQ ID NO:231).Optionally, the PRO792 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA56352-1358 vector deposited on May 6, 1998 as ATCC 209846.

37. PRO866

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to mindin and spondin proteins, wherein the polypeptideis designated in the present application as “PRO866”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO866 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO866 polypeptidehaving amino acid residues 1 to 331 of FIG. 87 (SEQ ID NO:236), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO866 polypeptide having amino acid residuesabout 27 to 229 of FIG. 87 (SEQ ID NO:236), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA53971-1359 vector deposited on Apr. 7, 1998 as ATCC 209750 whichincludes the nucleotide sequence encoding PRO866.

In another embodiment, the invention provides isolated PRO866polypeptide. In particular, the invention provides isolated nativesequence PRO866 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 331 of FIG. 87 (SEQ ID NO:236).Another embodiment of the present invention is directed to PRO866polypeptides comprising amino acids about 27 to 331 of FIG. 87 (SEQ IDNO:236). Optionally, the PRO866 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA53971-1359 vector deposited on Apr. 7, 1998 as ATCC 209750.

38. PRO871

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to CyP-60, wherein the polypeptide is designated in thepresent application as “PRO871”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO871 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO871 polypeptidehaving amino acid residues 1 to 472 of FIG. 89 (SEQ ID NO:245), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO871 polypeptide having amino acid residuesabout 22 to 472 of FIG. 89 (SEQ ID NO:245), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA50919-1361 vector deposited on May 6, 1998 as ATCC 209848 whichincludes the nucleotide sequence encoding PRO871.

In another embodiment, the invention provides isolated PRO871polypeptide. In particular, the invention provides isolated nativesequence PRO871 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 472 of FIG. 89 (SEQ ID NO:245).An additional embodiment of the present invention is directed to PRO871polypeptides comprising amino acids about 22 to 472 of FIG. 89 (SEQ IDNO:245). Optionally, the PRO871 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA50919-1361 vector deposited on May 6, 1998 as ATCC 209848.

39. PRO873

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to carboxylesterase, wherein the polypeptide isdesignated in the present application as “PRO873”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO873 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO873 polypeptidehaving amino acid residues 1 to 545 of FIG. 91 (SEQ ID NO:254), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO873 polypeptide having amino acid residuesabout 30 to about 545 of FIG. 91 (SEQ ID NO:254), or is complementary tosuch encoding nucleic acid sequence, and remains stably bound to itunder at least moderate, and optionally, under high stringencyconditions. The isolated nucleic acid sequence may comprise the cDNAinsert of the DNA44179-1362 vector deposited on May 6, 1998 as ATCC209851 which includes the nucleotide sequence encoding PRO873.

In another embodiment, the invention provides isolated PRO873polypeptide. In particular, the invention provides isolated nativesequence PRO873 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 545 of FIG. 91 (SEQ ID NO:254).Additional embodiments of the present invention are directed to PRO873polypeptides comprising amino acids about 30 to about 545 of FIG. 91(SEQ ID NO:254). Optionally, the PRO873 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA44179-1362 vector deposited on May 6, 1998 as ATCC 209851.

40. PRO940

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to CD33 and OB binding protein-2, wherein thepolypeptide is designated in the present application as “PRO940”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO940 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO940 polypeptidehaving amino acid residues 1 to 544 of FIG. 93 (SEQ ID NO:259), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO940 polypeptide having amino acid residuesabout 16 to 544 of FIG. 93 (SEQ ID NO:259) or 1 or about 16 to X of FIG.93 (SEQ ID NO:259), where X is any amino acid from 394 to 403 of FIG. 93(SEQ ID NO:259), or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions. The isolated nucleic acidsequence may comprise the cDNA insert of the DNA54002-1367 vectordeposited on Apr. 7, 1998 as ATCC 209754 which includes the nucleotidesequence encoding PRO940.

In another embodiment, the invention provides isolated PRO940polypeptide. In particular, the invention provides isolated nativesequence PRO940 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 544 of FIG. 93 (SEQ ID NO:259).Other embodiments of the present invention are directed to PRO940polypeptides comprising amino acids about 16 to 544 of FIG. 93 (SEQ IDNO:259) or 1 or about 16 to X of FIG. 93 (SEQ ID NO:259), where X is anyamino acid from 394 to 403 of FIG. 93 (SEQ ID NO:259). Optionally, thePRO940 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA54002-1367 vectordeposited on Apr. 7, 1998 as ATCC 209754.

41. PRO941

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to a cadherin protein, wherein the polypeptide isdesignated in the present application as “PRO941”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO941 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO941 polypeptidehaving amino acid residues 1 to 772 of FIG. 95 (SEQ ID NO:264), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO941 polypeptide having amino acid residuesabout 22 to 772 of FIG. 95 (SEQ ID NO:264) or 1 or about 22 to X of FIG.95 (SEQ ID NO:264), where X is any amino acid from 592 to 601 of FIG. 95(SEQ ID NO:264), or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions. The isolated nucleic acidsequence may comprise the cDNA insert of the DNA53906-1368 vectordeposited on Apr. 7, 1998 as ATCC 209747 which includes the nucleotidesequence encoding PRO941.

In another embodiment, the invention provides isolated PRO941polypeptide. In particular, the invention provides isolated nativesequence PRO941 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 772 of FIG. 95 (SEQ ID NO:264).Additional embodiments of the present invention are directed to PRO941polypeptides which comprise amino acid about 21 to 772 of FIG. 95 (SEQID NO:264) or 1 or about 22 to X of FIG. 95 (SEQ ID NO:264), where X isany amino acid from 592 to 601 of FIG. 95 (SEQ ID NO:264). Optionally,the PRO941 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the DNA53906-1368 vectordeposited on Apr. 7, 1998 as ATCC 209747.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA6415 comprising the nucleotide sequence ofFIG. 96 (SEQ ID NO:265).

42. PRO944

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to Clostridium perfringens enterotoxin receptor (CPE-R),wherein the polypeptide is designated in the present application as“PRO944”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO944 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO944 polypeptidehaving amino acid residues 1 to 211 of FIG. 98 (SEQ ID NO:270), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO944 polypeptide having amino acid residuesabout 22 to 229 of FIG. 98 (SEQ ID NO:270) or amino acid 1 or about 22to X of FIG. 98 (SEQ ID NO:270) where X is any amino acid from 77 to 80of FIG. 98 (SEQ ID NO:270), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. The isolated nucleicacid sequence may comprise the cDNA insert of the DNA52185-1370 vectordeposited on May 14, 1998 as ATCC 209861 which includes the nucleotidesequence encoding PRO944.

In another embodiment, the invention provides isolated PRO944polypeptide. In particular, the invention provides isolated nativesequence PRO944 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 211 of FIG. 98 (SEQ ID NO:270).Additional embodiments of the present invention are directed to PRO944polypeptides comprising amino acids about 22 to 211 of FIG. 98 (SEQ IDNO:270) or amino acid 1 or about 22 to X of FIG. 98 (SEQ ID NO:270),where X is any amino acid from 77 to 86 of FIG. 98 (SEQ ID NO:270).Optionally, the PRO944 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA52185-1370 vector deposited on May 14, 1998 as ATCC 209861.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA14007 comprising the nucleotide sequenceof FIG. 99 (SEQ ID NO:271).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA12733 comprising the nucleotide sequenceof FIG. 100 (SEQ ID NO:272).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA12746 comprising the nucleotide sequenceof FIG. 101 (SEQ ID NO:273).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA12834 comprising the nucleotide sequenceof FIG. 102 (SEQ ID NO:274).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA12846 comprising the nucleotide sequenceof FIG. 103 (SEQ ID NO:275).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA13104 comprising the nucleotide sequenceof FIG. 104 (SEQ ID NO:276).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA13259 comprising the nucleotide sequenceof FIG. 105 (SEQ ID NO:277).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA13959 comprising the nucleotide sequenceof FIG. 106 (SEQ ID NO:278).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA13961 comprising the nucleotide sequenceof FIG. 107 (SEQ ID NO:279).

43. PRO983

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to a vesicle associated protein, VAP-33, wherein thepolypeptide is designated in the present application as “PRO983”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO983 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO983 polypeptidehaving amino acid residues 1 to 243 of FIG. 109 (SEQ ID NO:284), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO983 polypeptide having amino acid residue1 to X of FIG. 109 (SEQ ID NO:284) where X is any amino acid from 219 to228 of FIG. 109 (SEQ ID NO:284), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA53977-1371vector deposited on May 14, 1998 as ATCC 209862 which includes thenucleotide sequence encoding PRO983.

In another embodiment, the invention provides isolated PRO983polypeptide. In particular, the invention provides isolated nativesequence PRO983 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 243 of FIG. 109 (SEQ ID NO:284).Additional embodiments of the present invention are directed to PRO983polypeptides comprising amino acid 1 to X of FIG. 109 (SEQ ID NO:284),where Y is any amino acid from 219 to 228 of FIG. 109 (SEQ ID NO:284).Optionally, the PRO983 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA53977-1371 vector deposited on May 14, 1998 as ATCC 209862.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA17130 comprising the nucleotide sequenceof FIG. 110 (SEQ ID NO:285).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA23466 comprising the nucleotide sequenceof FIG. 111 (SEQ ID NO:286).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA26818 comprising the nucleotide sequenceof FIG. 112 (SEQ ID NO:287).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA37618 comprising the nucleotide sequenceof FIG. 113 (SEQ ID NO:288).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA41732 comprising the nucleotide sequenceof FIG. 114 (SEQ ID NO:289).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA45980 comprising the nucleotide sequenceof FIG. 115 (SEQ ID NO:290).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA46372 comprising the nucleotide sequenceof FIG. 116 (SEQ ID NO:291).

44. PRO1057

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to proteases, wherein the polypeptide is designated inthe present application as “PRO1057”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1057 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1057 polypeptidehaving amino acid residues 1 to 413 of FIG. 118 (SEQ ID NO:296), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO1057 polypeptide having amino acidresidues about 17 to 413 of FIG. 118 (SEQ ID NO:296), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA57253-1382 vector deposited on May 14, 1998 asATCC 209867 which includes the nucleotide sequence encoding PRO1057.

In another embodiment, the invention provides isolated PRO1057polypeptide. In particular, the invention provides isolated nativesequence PRO1057 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 413 of FIG. 118 (SEQ ID NO:296).Additional embodiments of the present invention are directed to PRO1057polypeptides comprising amino acids about 17 to 413 of FIG. 118 (SEQ IDNO:296). Optionally, the PRO1057 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA57253-1382 vector deposited on May 14, 1998 as ATCC 209867.

45. PRO1071

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to thrombospondin, wherein the polypeptide is designatedin the present application as “PRO1071”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1071 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1071 polypeptidehaving amino acid residues 1 to 525 of FIG. 120 (SEQ ID NO:301), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO1071 polypeptide having amino acidresidues about 26 to 525 of FIG. 120 (SEQ ID NO:301), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA58847-1383 vector deposited on May 20, 1998 asATCC 209879 which includes the nucleotide sequence encoding PRO1071.

In another embodiment, the invention provides isolated PRO1071polypeptide. In particular, the invention provides isolated nativesequence PRO1071 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 525 of FIG. 120 (SEQ ID NO:301).Additional embodiments of the present invention are directed to PRO1071polypeptides comprising amino acids about 26 to 525 of FIG. 120 (SEQ IDNO:301). Optionally, the PRO1071 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA58847-1383 vector deposited on May 20, 1998 as ATCC 209879.

46. PRO1072

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to reductase proteins, wherein the polypeptide isdesignated in the present application as “PRO1072”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1072 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1072 polypeptidehaving amino acid residues 1 to 336 of FIG. 122 (SEQ ID NO:303), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO1072 polypeptide having amino acidresidues about 22 to 336 of FIG. 122 (SEQ ID NO:303), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA58747-1384 vector deposited on May 14, 1998 asATCC 209868 which includes the nucleotide sequence encoding PRO1072.

In another embodiment, the invention provides isolated PRO1072polypeptide. In particular, the invention provides isolated nativesequence PRO1072 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 336 of FIG. 122 (SEQ ID NO:303).Additional embodiments of the present invention are directed to PRO1072polypeptides comprising amino acids about 22 to 336 of FIG. 122 (SEQ IDNO:303). Optionally, the PRO1072 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA58747-1384 vector deposited on May 14, 1998 as ATCC 209868.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA40210 comprising the nucleotide sequenceof FIG. 123 (SEQ ID NO:304).

47. PRO1075

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to protein disulfide isomerase, wherein the polypeptideis designated in the present application as “PRO1075”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1075 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1075 polypeptidehaving amino acid residues 1 to 406 of FIG. 125 (SEQ ID NO:309), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO1075 polypeptide having amino acidresidues about 30 to 406 of FIG. 125 (SEQ ID NO:309), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the DNA57689-1385 vector deposited on May 14, 1998 asATCC 209869 which includes the nucleotide sequence encoding PRO1075.

In another embodiment, the invention provides isolated PRO1075polypeptide. In particular, the invention provides isolated nativesequence PRO1075 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 406 of FIG. 125 (SEQ ID NO:309).Additional embodiments of the present invention are directed to PRO1075polypeptides comprising amino acids about 30 to 406 of FIG. 125 (SEQ IDNO:309). Optionally, the PRO1075 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA57689-1385 vector deposited on May 14, 1998 as ATCC 209869.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA13059 comprising the nucleotide sequenceof FIG. 126 (SEQ ID NO:310).

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA19463 comprising the nucleotide sequenceof FIG. 127 (SEQ ID NO:311).

48. PRO181

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the cornichon protein, wherein the polypeptide isdesignated in the present application as “PRO181”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO181 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO181 polypeptidehaving amino acid residues 1 to 144 of FIG. 129 (SEQ ID NO:322), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO181 polypeptide having amino acid residuesabout 21 to 144 of FIG. 129 (SEQ ID NO:322) or amino acid 1 or about 21to X of FIG. 129 (SEQ ID NO:322) where X is any amino acid from 52 to 61of FIG. 129 (SEQ ID NO:322), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA23330-1390vector deposited on Apr. 14, 1998 as ATCC 209775 which includes thenucleotide sequence encoding PRO181.

In another embodiment, the invention provides isolated PRO181polypeptide. In particular, the invention provides isolated nativesequence PRO181 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 144 of FIG. 129 (SEQ ID NO:322).Additional embodiments of the present invention are directed to PRO181polypeptides comprising amino acids about 21 to 144 of FIG. 129 (SEQ IDNO:322) or amino acid 1 or about 21 to X of FIG. 129 (SEQ ID NO:322),where X is any amino acid from 52 to 61 of FIG. 129 (SEQ ID NO:322).Optionally, the PRO181 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA23330-1390 vector deposited on Apr. 14, 1998 as ATCC 209775.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA13242 comprising the nucleotide sequenceof FIG. 130 (SEQ ID NO:323).

49. PRO195

Applicants have identified a cDNA clone that encodes a noveltransmembrane polypeptide, wherein the polypeptide is designated in thepresent application as “PRO195”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO195 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO195 polypeptidehaving amino acid residues 1 to 323 of FIG. 132 (SEQ ID NO:330), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO195 polypeptide having amino acid residuesabout 32 to 323 of FIG. 132 (SEQ ID NO:330) or amino acid 1 or about 32to X of FIG. 132 (SEQ ID NO:330) where X is any amino acid from 236 to245 of FIG. 132 (SEQ ID NO:330), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA26847-1395vector deposited on Apr. 14, 1998 as ATCC 209772 which includes thenucleotide sequence encoding PRO195.

In another embodiment, the invention provides isolated PRO195polypeptide. In particular, the invention provides isolated nativesequence PRO195 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 323 of FIG. 132 (SEQ ID NO:330).Additional embodiments of the present invention are directed to PRO195polypeptides comprising amino acids about 32 to 323 of FIG. 132 (SEQ IDNO:330) or amino acid 1 or about 32 to X of FIG. 132 (SEQ ID NO:330),where X is any amino acid from 236 to 245 of FIG. 132 (SEQ ID NO:330).Optionally, the PRO195 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA26847-1395 vector deposited on Apr. 14, 1998 as ATCC 209772.

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of FIG. 133 (SEQ ID NO:331),herein designated DNA15062.

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of FIG. 134 (SEQ ID NO:332),herein designated DNA13199.

50. PRO865

Applicants have identified a cDNA clone that encodes a novel secretedpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO865”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO865 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO865 polypeptidehaving amino acid residues 1 to 468 of FIG. 136 (SEQ ID NO:337), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO865 polypeptide having amino acid residuesabout 24 to 229 of FIG. 136 (SEQ ID NO:337), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA53974-1401 vector deposited on Apr. 14, 1998 as ATCC 209774 whichincludes the nucleotide sequence encoding PRO865.

In another embodiment, the invention provides isolated PRO865polypeptide. In particular, the invention provides isolated nativesequence PRO865 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 468 of FIG. 136 (SEQ ID NO:337).An additional embodiment of the present invention is directed to aPRO865 polypeptide comprising amino acids about 24 to 468 of FIG. 136(SEQ ID NO:337). Optionally, the PRO865 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA53974-1401 vector deposited on Apr. 14, 1998 as ATCC 209774.

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of FIG. 137 (SEQ ID NO:338),herein designated as DNA37642.

51. PRO827

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to integrin proteins, wherein the polypeptide isdesignated in the present application as “PRO827”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO827 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO827 polypeptidehaving amino acid residues 1 to 124 of FIG. 139 (SEQ ID NO:346), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO827 polypeptide having amino acid residuesabout 23 to 124 of FIG. 139 (SEQ ID NO:346), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA57039-1402 vector deposited on Apr. 14, 1998 as ATCC 209777 whichincludes the nucleotide sequence encoding PRO827.

In another embodiment, the invention provides isolated PRO827polypeptide. In particular, the invention provides isolated nativesequence PRO827 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 124 of FIG. 139 (SEQ ID NO:346).An additional embodiment of the present invention is directed to aPRO827 polypeptide comprising amino acids about 23 to 124 of FIG. 139(SEQ ID NO:346). Optionally, the PRO827 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe DNA57039-1402 vector deposited on Apr. 14, 1998 as ATCC 209777.

52. PRO1114

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to cytokine receptor family-4 proteins, wherein thepolypeptide is designated in the present application as “PRO1114”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1114 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1114 polypeptidehaving amino acid residues 1 to 311 of FIG. 142 (SEQ ID NO:352), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO1114 polypeptide having amino acidresidues about 30 to 311 of FIG. 142 (SEQ ID NO:352) or amino acid 1 orabout 30 to X of FIG. 142 (SEQ ID NO:352), where X is any amino acidfrom 225 to 234 of FIG. 142 (SEQ ID NO:352), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA57033-1403 vector deposited on May 27, 1998 as ATCC 209905 whichincludes the nucleotide sequence encoding PRO1114.

In another embodiment, the invention provides isolated PRO1114polypeptide. In particular, the invention provides isolated nativesequence PRO1114 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 311 of FIG. 142 (SEQ ID NO:352).Additional embodiments of the present invention are directed to PRO114polypeptides comprising amino acids about 30 to 311 of FIG. 142 (SEQ IDNO:352) or amino acid 1 or about 30 to X of FIG. 142 (SEQ ID NO:352),where X is any amino acid from 225 to 234 of FIG. 142 (SEQ ID NO:352).Optionally, the PRO1114 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA57033-1403 vector deposited on May 27, 1998 as ATCC 209905.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA48466 comprising the nucleotide sequenceof FIG. 143 (SEQ ID NO:353).

A cDNA clone (DNA57033-1403) has been identified that encodes a novelinterferon receptor polypeptide, designated in the present applicationas “PRO1114 interferon receptor”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1114 interferon receptorpolypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a PRO1114 interferon receptor polypeptide having the sequenceof amino acid residues from about 1 or about 30 to about 311, inclusiveof FIG. 142 (SEQ ID NO:352), or (b) the complement of the DNA moleculeof (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1114 interferon receptor polypeptide comprisingDNA hybridizing to the complement of the nucleic acid between aboutnucleotides 250 or about 337 and about 1182, inclusive, of FIG. 141 (SEQID NO:351). Preferably, hybridization occurs under stringenthybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the human protein cDNA in ATCC Deposit No. 209905(DNA57033-1403) or (b) the complement of the nucleic acid molecule of(a). In a preferred embodiment, the nucleic acid comprises a DNAencoding the same mature polypeptide encoded by the human protein cDNAin ATCC Deposit No. 209905 (DNA57033-1403).

In still a further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues 1 or about 30 to about 311, inclusive of FIG. 142 (SEQ IDNO:352), or (b) the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least 10 nucleotides and produced by hybridizing atest DNA molecule under stringent conditions with (a) a DNA moleculeencoding a PRO1114 interferon receptor polypeptide having the sequenceof amino acid residues from 1 or about 30 to about 311, inclusive ofFIG. 142 (SEQ ID NO:352), or (b) the complement of the DNA molecule of(a), and, if the DNA molecule has at least about an 80% sequenceidentity, prefereably at least about an 85% sequence identity, morepreferably at least about a 90% sequence identity, most preferably atleast about a 95% sequence identity to (a) or (b), isolating the testDNA molecule.

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1114 interferon receptorpolypeptide, with or without the N-terminal signal sequence and/or theinitiating methionine, and its soluble, i.e., transmembrane domaindeleted or inactivated variants, or is complementary to such encodingnucleic acid molecule. The signal peptide has been tentativelyidentified as extending from about amino acid position 1 to about aminoacid position 29 in the sequence of FIG. 142 (SEQ ID NO:352). Thetransmembrane domain has been tentatively identified as extending fromabout amino acid position 230 to about amino acid position 255 in thePRO114 interferon receptor amino acid sequence (FIG. 142, SEQ IDNO:352).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 30 to about 311, inclusive of FIG. 142 (SEQ ID NO:352), or (b)the complement of the DNA of (a).

Another embodiment is directed to fragments of a PRO1114 interferonreceptor polypeptide coding sequence that may find use as hybridizationprobes. Such nucleic acid fragments are from about 20 to about 80nucleotides in length, preferably from about 20 to about 60 nucleotidesin length, more preferably from about 20 to about 50 nucleotides inlength and most preferably from about 20 to about 40 nucleotides inlength and may be derived from the nucleotide sequence shown in FIG. 141(SEQ ID NO:351).

In another embodiment, the invention provides a vector comprising DNAencoding PRO1114 interferon receptor or its variants. The vector maycomprise any of the isolated nucleic acid molecules hereinaboveidentified.

A host cell comprising such a vector is also provided. By way ofexample, the host cells may be CHO cells, E. coli, or yeast. A processfor producing PRO1114 interferon receptor polypeptides is furtherprovided and comprises culturing host cells under conditions suitablefor expression of PRO1114 interferon receptor and recovering PRO1114interferon receptor from the cell culture.

In another embodiment, the invention provides isolated PRO1114interferon receptor polypeptide encoded by any of the isolated nucleicacid sequences hereinabove identified.

In a specific aspect, the invention provides isolated native sequencePRO1114 interferon receptor polypeptide, which in certain embodiments,includes an amino acid sequence comprising residues 1 or about 30 toabout 311 of FIG. 142 (SEQ ID NO:352).

In another aspect, the invention concerns an isolated PRO1114 interferonreceptor polypeptide, comprising an amino acid sequence having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues 1 or about 30 to about 311, inclusive of FIG. 142 (SEQ IDNO:352).

In a further aspect, the invention concerns an isolated PRO1114interferon receptor polypeptide, comprising an amino acid sequencescoring at least about 80% positives, preferably at least about 85%positives, more preferably at least about 90% positives, most preferablyat least about 95% positives when compared with the amino acid sequenceof residues 1 or about 30 to about 311, inclusive of FIG. 142 (SEQ IDNO:352).

In yet another aspect, the invention concerns an isolated PRO1114interferon receptor polypeptide, comprising the sequence of amino acidresidues 1 or about 30 to about 311, inclusive of FIG. 142 (SEQ IDNO:352), or a fragment thereof sufficient to provide a binding site foran anti-PRO1114 interferon receptor antibody. Preferably, the PRO1114interferon receptor fragment retains a qualitative biological activityof a native PRO1114 interferon receptor polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO1114 interferon receptor polypeptidehaving the sequence of amino acid residues from about 1 or about 30 toabout 311, inclusive of FIG. 142 (SEQ ID NO:352), or (b) the complementof the DNA molecule of (a), and if the test DNA molecule has at leastabout an 80% sequence identity, preferably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), (ii) culturing a host cell comprising the test DNA moleculeunder conditions suitable for expression of the polypeptide, and (iii)recovering the polypeptide from the cell culture.

In another embodiment, the invention provides chimeric moleculescomprising a PRO1114 interferon receptor polypeptide fused to aheterologous polypeptide or amino acid sequence. An example of such achimeric molecule comprises a PRO114 interferon receptor polypeptidefused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to a PRO1114 interferon receptor polypeptide.Optionally, the antibody is a monoclonal antibody.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1114 interferon receptor polypeptide. In aparticular embodiment, the agonist or antagonist is an anti-PRO1114interferon receptor antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native PRO1114 interferon receptorpolypeptide by contacting the native PRO1114 interferon receptorpolypeptide with a candidate molecule and monitoring a biologicalactivity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising a PRO1114 interferon receptor polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

53. PRO237

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to carbonic anhydrase, wherein the polypeptide isdesignated in the present application as “PRO237”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO237 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO237 polypeptidehaving amino acid residues 1 to 328 of FIG. 145 (SEQ ID NO:358), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO237 polypeptide having amino acid residuesabout 24 to 328 of FIG. 145 (SEQ ID NO:358) or amino acid 1 or about 24to X of FIG. 145 (SEQ ID NO:358), where X is any amino acid from 172 to181 of FIG. 145 (SEQ ID NO:358), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA34353-1428vector deposited on May 12, 1998 as ATCC 209855 which includes thenucleotide sequence encoding PRO237.

In another embodiment, the invention provides isolated PRO237polypeptide. In particular, the invention provides isolated nativesequence PRO237 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 328 of FIG. 145 (SEQ ID NO:358).Additional embodiments of the present invention are directed to PRO237polypeptides comprising amino acids about 24 to 328 of FIG. 145 (SEQ IDNO:358) or amino acid 1 or about 24 to X of FIG. 145 (SEQ ID NO:358),where X is any amino acid from 172 to 181 of FIG. 145 (SEQ ID NO:358).Optionally, the PRO237 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA34353-1428 vector deposited on May 12, 1998 as ATCC 209855.

54. PRO541

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to a trypsin inhibitor protein, wherein the polypeptideis designated in the present application as “PRO541”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO541 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO541 polypeptidehaving amino acid residues 1 to 500 of FIG. 147 (SEQ ID NO:363), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO541 polypeptide having amino acid residuesabout 21 to 500 of FIG. 147 (SEQ ID NO:363), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of theDNA45417-1432 vector deposited on May 27, 1998 as ATCC 209910 whichincludes the nucleotide sequence encoding PRO541.

In another embodiment, the invention provides isolated PRO541polypeptide. In particular, the invention provides isolated nativesequence PRO541 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 500 of FIG. 147 (SEQ ID NO:363).Additional embodiments of the present invention are directed to PRO541polypeptides comprising amino acids about 21 to 500 of FIG. 147 (SEQ IDNO:363). Optionally, the PRO541 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA45417-1432 vector deposited on May 27, 1998 as ATCC 209910.

55. PRO273

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO273”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO273 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO273 polypeptidehaving amino acid residues 1 through 111 of FIG. 149 (SEQ ID NO:370), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO273polypeptide. In particular, the invention provides isolated nativesequence PRO273 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 111 of FIG. 149 (SEQ IDNO:370).

56. PRO701

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to neuroligins 1, 2, and 3, wherein the polypeptide isdesignated in the present application as “PRO701”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO701 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO701 polypeptidehaving amino acid residues 1 through 816 of FIG. 151 (SEQ ID NO:375), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited with the ATCC on Mar. 31, 1998which includes the nucleotide sequence encoding PRO701.

In another embodiment, the invention provides isolated PRO701polypeptide. In particular, the invention provides isolated nativesequence PRO701 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 816 of FIG. 151 (SEQ IDNO:375). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO701 polypeptide. Optionally,the PRO701 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited with theATCC on Mar. 31, 1998.

57. PRO704

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with VIP36, wherein the polypeptide isdesignated in the present application as “PRO704”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO704 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO704 polypeptidehaving amino acid residues 1 through 348 of FIG. 153 (SEQ ID NO:380), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Mar. 31, 1998 with the ATCCas DNA50911-1288, which includes the nucleotide sequence encodingPRO704.

In another embodiment, the invention provides isolated PRO704polypeptide. In particular, the invention provides isolated nativesequence PRO704 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 348 of FIG. 153 (SEQ IDNO:380). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO704 polypeptide. Optionally,the PRO704 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Mar.31, 1998 with the ATCC as DNA50911-1288.

58. PRO706

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to prostatic acid phosphatase precursor and lysosomalacid phosphatase precursor, wherein the polypeptide is designated in thepresent application as “PRO706”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO706 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO706 polypeptidehaving amino acid residues 1 through 480 of FIG. 155 (SEQ ID NO:385), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Apr. 21, 1998 with the ATCCas DNA48329-1290 which includes the nucleotide sequence encoding PRO706.

In another embodiment, the invention provides isolated PRO706polypeptide. In particular, the invention provides isolated nativesequence PRO706 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 480 of FIG. 155 (SEQ IDNO:385), or comprising residues 19 through 480 of FIG. 155 (SEQ IDNO:385). Optionally, the PRO706 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of the vectordeposited on Apr. 21, 1998 with the ATCC as DNA48329-1290.

59. PRO707

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to cadherins, particularly cadherin FIB3, wherein thepolypeptide is designated in the present application as “PRO707”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO707 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO707 polypeptidehaving amino acid residues 1 to 916 of FIG. 157 (SEQ ID NO:390), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 27, 1998 with the ATCC asDNA48306-1291 which includes the nucleotide sequence encoding PRO707.

In another embodiment, the invention provides isolated PRO707polypeptide. In particular, the invention provides isolated nativesequence PRO707 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 916 of FIG. 157 (SEQ ID NO:390).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO707 polypeptide. Optionally, thePRO707 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on May27, 1998 with the ATCC as DNA48306-1291.

60. PRO322

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to neuropsin, wherein the polypeptide is designated inthe present application as “PRO322”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO322 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO322 polypeptidehaving amino acid residues 1 or 24 through 260 of FIG. 159 (SEQ IDNO:395), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. The isolated nucleic acid sequence maycomprise the cDNA insert of the vector deposited on Mar. 11, 1998 asATCC no. 209669 which includes the nucleotide sequence encoding PRO322.

In another embodiment, the invention provides isolated PRO322polypeptide. In particular, the invention provides isolated nativesequence PRO322 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 or 24 through 260 of FIG. 159 (SEQID NO:395). An additional embodiment of the present invention isdirected to an isolated extracellular domain of a PRO322 polypeptide.Optionally, the PRO322 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of the vectordeposited on Mar. 11, 1998 as ATCC no. 209669.

61. PRO526

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with ALS, wherein the polypeptide is designatedin the present application as “PRO526”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO526 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO526 polypeptidehaving amino acid residues 1 to 473 of FIG. 161 (SEQ ID NO:400), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Mar. 26, 1998 with the ATCCas DNA44184-1319 which includes the nucleotide sequence encoding PRO526.

In another embodiment, the invention provides isolated PRO526polypeptide. In particular, the invention provides isolated nativesequence PRO526 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 473 of FIG. 161 (SEQ ID NO:400).Optionally, the PRO526 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of the vectordeposited on Mar. 26, 1998 with the ATCC as DNA44184-1319 which includesthe nucleotide sequence encoding PRO526.

62. PRO531

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with protocadherins, wherein the polypeptide isdesignated in the present application as “PRO531”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO531 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO531 polypeptidehaving amino acid residues 1 to 789 of FIG. 163 (SEQ ID NO:405), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Mar. 26, 1998 asDNA48314-1320 which includes the nucleotide sequence encoding PRO531.

In another embodiment, the invention provides isolated PRO531polypeptide. In particular, the invention provides isolated nativesequence PRO531 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 789 of FIG. 163 (SEQ ID NO:405).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO531 polypeptide. Optionally, thePRO531 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Mar.26, 1998 as DNA48314-1320.

63. PRO534

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with disulfide isomerase (sometimes referred toherein as protein disulfide isomerase), wherein the polypeptide isdesignated in the present application as “PRO534”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO534 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO534 polypeptidehaving amino acid residues 1 to 360 of FIG. 165 (SEQ ID NO:410), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Mar. 26, 1998 asDNA48333-1321 which includes the nucleotide sequence encoding PRO534.

In another embodiment, the invention provides isolated PRO534polypeptide. In particular, the invention provides isolated nativesequence PRO534 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 360 of FIG. 165 (SEQ ID NO:410).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO534 polypeptide. Optionally, thePRO534 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Mar.26, 1998 as DNA48333-1321.

64. PRO697

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with sFRPs, wherein the polypeptide isdesignated in the present application as “PRO697”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO697 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO697 polypeptidehaving amino acid residues 1 through 295 of FIG. 167 (SEQ ID NO:415), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited with the ATCC on Mar. 26, 1998as DNA50920-1325 which includes the nucleotide sequence encoding PRO697.

In another embodiment, the invention provides isolated PRO697polypeptide. In particular, the invention provides isolated nativesequence PRO697 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 295 of FIG. 167 (SEQ IDNO:415). Optionally, the PRO697 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of the vectordeposited with the ATCC on Mar. 26, 1998 as DNA50920-1325.

65. PRO717

Applicants have identified a cDNA clone that encodes a novel 12transmembrane polypeptide, wherein the polypeptide is designated in thepresent application as “PRO717”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO717 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO717 polypeptidehaving amino acid residues 1 through 560 of FIG. 169 (SEQ ID NO:420), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Apr. 28, 1998 with the ATCCas DNA50988-1326 which includes the nucleotide sequence encoding PRO717.

In another embodiment, the invention provides isolated PRO717polypeptide. In particular, the invention provides isolated nativesequence PRO717 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 560 of FIG. 169 (SEQ IDNO:420). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO717 polypeptide. Optionally,the PRO717 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Apr.28, 1998 with the ATCC as DNA50988-1326.

66. PRO731

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with protocadherin 4, wherein the polypeptideis designated in the present application as “PRO731”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO731 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO731 polypeptidehaving amino acid residues 1 through 1184 of FIG. 171 (SEQ ID NO:425),or is complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Mar. 31, 1998 with the ATCCas DNA48331-1329 which includes the nucleotide sequence encoding PRO731.

In another embodiment, the invention provides isolated PRO731polypeptide. In particular, the invention provides isolated nativesequence PRO731 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 1184 of FIG. 171 (SEQ IDNO:425). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO731 polypeptide. Optionally,the PRO731 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Mar.31, 1998 with the ATCC as DNA48331-1329.

67. PRO218

Applicants have identified a cDNA clone that encodes a novelmulti-transmembrane protein having sequence identity with membraneregulator proteins, wherein the polypeptide is designated in the presentapplication as “PRO218”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO218 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO218 polypeptidehaving amino acid residues 1 through 455 of FIG. 173 (SEQ ID NO:430), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Apr. 28, 1998 with the ATCCas DNA30867-1335 which includes the nucleotide sequence encoding PRO218.

In another embodiment, the invention provides isolated PRO218polypeptide. In particular, the invention provides isolated nativesequence PRO218 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 455 of FIG. 173 (SEQ IDNO:430). Optionally, the PRO218 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of the vectordeposited on Apr. 28, 1998 with the ATCC as DNA30867-1335.

In another embodiment, the invention provides an expressed sequence tag(EST) sequence comprising the nucleotide sequence of FIG. 174 (SEQ IDNO:431), designated herein as DNA14472.

In another embodiment, the invention provides an expressed sequence tag(EST) sequence comprising the nucleotide sequence of FIG. 175 (SEQ IDNO:432), designated herein as DNA15846.

68. PRO768

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with integrins, wherein the polypeptide isdesignated in the present application as “PRO768”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO768 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO768 polypeptidehaving amino acid residues 1 through 1141 of FIG. 177 (SEQ ID NO:437),or is complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Apr. 6, 1998 as DNA55737-1345which includes the nucleotide sequence encoding PRO768.

In another embodiment, the invention provides isolated PRO768polypeptide. In particular, the invention provides isolated nativesequence PRO768 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 1141 of FIG. 177 (SEQ IDNO:437). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO768 polypeptide. Optionally,the PRO768 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Apr.6, 1998 as DNA55737-1345.

69. PRO771

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with testican, wherein the polypeptide isdesignated in the present application as “PRO771”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO771 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO771 polypeptidehaving amino acid residues 1 through 436 of FIG. 179 (SEQ ID NO:442), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Apr. 7, 1998 with the ATCC asDNA49829-1346 which includes the nucleotide sequence encoding PRO771.

In another embodiment, the invention provides isolated PRO771polypeptide. In particular, the invention provides isolated nativesequence PRO771 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 436 of FIG. 179 (SEQ IDNO:442). Optionally, the PRO771 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of the vectordeposited on Apr. 7, 1998 with the ATCC as DNA49829-1346.

70. PRO733

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with the T1I/ST2 receptor binding protein,wherein the polypeptide is designated in the present application as“PRO733”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO733 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO733 polypeptidehaving amino acid residues 1 through 229 of FIG. 181 (SEQ ID NO:447), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Apr. 7, 1998 with the ATCC asDNA52196-1348 which includes the nucleotide sequence encoding PRO733.

In another embodiment, the invention provides isolated PRO733polypeptide. In particular, the invention provides isolated nativesequence PRO733 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 229 of FIG. 181 (SEQ IDNO:447). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO733 polypeptide. Optionally,the PRO733 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on Apr.7, 1998 as DNA52196-1348.

71. PRO162

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with pancreatitis-associated protein, whereinthe polypeptide is designated in the present application as “PRO162”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO162 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO162 polypeptidehaving amino acid residues 1 through 175 of FIG. 183 (SEQ ID NO:452), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 6, 1998 with the ATCC asDNA56965-1356 which includes the nucleotide sequence encoding PRO162.

In another embodiment, the invention provides isolated PRO162polypeptide. In particular, the invention provides isolated nativesequence PRO162 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 175 of FIG. 183 (SEQ IDNO:452). Optionally, the PRO162 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of the vectordeposited on May 6, 1998 with the ATCC as DNA56965-1356.

72. PRO788

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with anti-neoplastic urinary protein, whereinthe polypeptide is designated in the present application as “PRO788”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO788 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO788 polypeptidehaving amino acid residues 1 through 125 of FIG. 185 (SEQ ID NO:454), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 6, 1998 with the ATCC asDNA56405-1357 which includes the nucleotide sequence encoding PRO788.

In another embodiment, the invention provides isolated PRO788polypeptide. In particular, the invention provides isolated nativesequence PRO788 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 125 of FIG. 185 (SEQ IDNO:454). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO788 polypeptide. Optionally,the PRO788 polypeptide is obtained or is obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited on May 6,1998 with the ATCC as DNA56405-1357.

73. PRO1008

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with dickkopf-1 (dkk-1), wherein thepolypeptide is designated in the present application as “PRO1008”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1008 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1008 polypeptidehaving amino acid residues 1 through 266 of FIG. 187 (SEQ ID NO:456), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 20, 1998 with the ATCC asDNA57530-1375 which includes the nucleotide sequence encoding PRO1008.

In another embodiment, the invention provides isolated PRO1008polypeptide. In particular, the invention provides isolated nativesequence PRO1008 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 266 of FIG. 187 (SEQ IDNO:456). Optionally, the PRO1008 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe vector deposited on May 20, 1998 with the ATCC as DNA57530-1375.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA16508 comprising the nucleotide sequenceof FIG. 188 (SEQ ID NO:457).

74. PRO1012

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with disulfide isomerase and phospholipase C,wherein the polypeptide is designated in the present application as“PRO1012”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1012 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1012 polypeptidehaving amino acid residues 1 through 747 of FIG. 190 (SEQ ID NO:459), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 14, 1998 with the ATCC asDNA56439-1376, which includes the nucleotide sequence encoding PRO1012.

In another embodiment, the invention provides isolated PRO1012polypeptide. In particular, the invention provides isolated nativesequence PRO1012 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 747 of FIG. 190 (SEQ IDNO:459). Optionally, the PRO1012 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe vector deposited on May 14, 1998 with the ATCC as DNA56439-1376.

75. PRO1014

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with reductase, wherein the polypeptide isdesignated in the present application as “PRO1014”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1014 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1014 polypeptidehaving amino acid residues 1 through 300 of FIG. 192 (SEQ ID NO:464), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 20, 1998 as DNA56409-1377with the ATCC which includes the nucleotide sequence encoding PRO1014.

In another embodiment, the invention provides isolated PRO1014polypeptide. In particular, the invention provides isolated nativesequence PRO1014 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 300 of FIG. 192 (SEQ IDNO:464). Optionally, the PRO1014 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe vector deposited on May 20, 1998 as DNA56409-1377 with the ATCC.

76. PRO1017

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with HNK-1 sulfotransferase, wherein thepolypeptide is designated in the present application as “PRO1017”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1017 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1017 polypeptidehaving amino acid residues 1 through 414 of FIG. 194 (SEQ ID NO:466), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 20, 1998 with the ATCC asDNA56112-1379 which includes the nucleotide sequence encoding PRO1017.

In another embodiment, the invention provides isolated PRO1017polypeptide. In particular, the invention provides isolated nativesequence PRO1017 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 414 of FIG. 194 (SEQ IDNO:466). Optionally, the PRO1017 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe vector deposited on May 20, 1998 with the ATCC as DNA56112-1379.

77. PRO474

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with dehydrogenase, wherein the polypeptide isdesignated in the present application as “PRO474”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO474 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO474 polypeptidehaving amino acid residues 1 through 270 of FIG. 196 (SEQ ID NO:468), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 14, 1998 with the ATCC asDNA56045-1380 which includes the nucleotide sequence encoding PRO474.

In another embodiment, the invention provides isolated PRO474polypeptide. In particular, the invention provides isolated nativesequence PRO474 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 270 of FIG. 196 (SEQ IDNO:468). Optionally, the PRO474 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of the vectordeposited on May 14, 1998 with the ATCC as DNA56045-1380.

78. PRO1031

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with IL-17, wherein the polypeptide isdesignated in the present application as “PRO1031”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1031 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1031 polypeptidehaving amino acid residues 1 through 180 of FIG. 198 (SEQ ID NO:470), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 14, 1998 with the ATCC asDNA59294-1381 which includes the nucleotide sequence encoding PRO1031.

In another embodiment, the invention provides isolated PRO1031polypeptide. In particular, the invention provides isolated nativesequence PRO1031 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 180 of FIG. 198 (SEQ IDNO:470). Optionally, the PRO1031 polypeptide is obtained or isobtainable by expressing the polypeptide encoded by the cDNA insert ofthe vector deposited on May 14, 1998 with the ATCC as DNA59294-1381.

79. PRO938

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity to protein disulfide isomerase, wherein thepolypeptide is designated in the present application as “PRO938”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO938 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO938 polypeptidehaving amino acid residues 1 to 349 of FIG. 200 (SEQ ID NO:472), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In other aspects, the isolated nucleic acidcomprises DNA encoding the PRO938 polypeptide having amino acid residuesabout 23 to 349 of FIG. 200 (SEQ ID NO:472) or amino acid 1 or about 23to X of FIG. 200 (SEQ ID NO:472), where X is any amino acid from 186 to195 of FIG. 200 (SEQ ID NO:472), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. The isolatednucleic acid sequence may comprise the cDNA insert of the DNA56433-1406vector deposited on May 12, 1998, as ATCC Accession No. 209857 whichincludes the nucleotide sequence encoding PRO938.

In another embodiment, the invention provides isolated PRO938polypeptide. In particular, the invention provides isolated nativesequence PRO938 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 349 of FIG. 200 (SEQ ID NO:472).Additional embodiments of the present invention are directed to PRO938polypeptides comprising amino acids about 23 to 349 of FIG. 200 (SEQ IDNO:472) or amino acid 1 or about 23 to X of FIG. 200 (SEQ ID NO:472),where X is any amino acid from 186 to 195 of FIG. 200 (SEQ ID NO:472).Optionally, the PRO938 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of theDNA56433-1406 vector deposited on May 12, 1998, as ATCC Accession No.209857.

80. PRO1082

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with a lectin-like oxidized LDL receptor,wherein the polypeptide is designated in the present application as“PRO1082”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1082 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1082 polypeptidehaving amino acid residues 1 through 201 of FIG. 202 (SEQ ID NO:477), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 14, 1998 with the ATCC asDNA53912-1457 which includes the nucleotide sequence encoding PRO1082.

In another embodiment, the invention provides isolated PRO1082polypeptide. In particular, the invention provides isolated nativesequence PRO1082 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 201 of FIG. 202 (SEQ IDNO:477). An additional embodiment of the present invention is directedto an isolated domain of a PRO1082 polypeptide, excluding thetransmembrane domain. Optionally, the PRO1082 polypeptide is obtained oris obtainable by expressing the polypeptide encoded by the cDNA insertof the vector deposited on May 14, 1998 with the ATCC as DNA53912-1457.

81. PRO1083

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with a 7TM receptor, latrophilin-relatedprotein 1, and a macrophage restricted cell surface glycoprotein,wherein the polypeptide is designated in the present application as“PRO1083”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1083 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO1083 polypeptidehaving amino acid residues 1 through 693 of FIG. 204 (SEQ ID NO:483), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on May 12, 1998 with the ATCC asDNA50921-1458 which includes the nucleotide sequence encoding PRO1083.

In another embodiment, the invention provides isolated PRO1083polypeptide. In particular, the invention provides isolated nativesequence PRO1083 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 693 of FIG. 204 (SEQ IDNO:483). An additional embodiment of the present invention is directedto an isolated extracellular domain of a PRO1083 polypeptide.Optionally, the PRO1083 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of the vectordeposited on May 12, 1998 with the ATCC as DNA50921-1458.

In another embodiment, the invention provides an expressed sequence tag(EST) designated herein as DNA24256 which comprises the nucleotidesequence of FIG. 205 (SEQ ID NO:484).

82. PRO200

The objects of this invention, as defined generally supra, are achievedat least in part by the provision of a novel polypeptide, VEGF-E alsoherein designated PRO200, (SEQ ID NO:488) and the nucleic acid encodingtherefor, SEQ ID NO:487, residues 259 through 1293.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a VEGF-E polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the VEGF-E polypeptidehaving amino acid residues 1 through 345 of FIG. 207 (SEQ ID NO:488), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under low stringency conditions. In anotherembodiment, variants are provided wherein the VEGF-E nucleic acid hassingle or multiple deletions, substitutions, insertions, truncations orcombinations thereof.

In another embodiment, the invention provides isolated VEGF-Epolypeptide. In particular, the invention provides an isolated nativesequence VEGF-E polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 345 of FIG. 207 (SEQ IDNO:488). In another embodiment, variants are provided wherein the VEGF-Epolypeptide has single or multiple deletions, substitutions, insertions,truncations or combinations thereof.

In yet further embodiments, the present invention is directed tocompositions useful for treating indications where proliferation,survival and/or differentiation of cells is desired, comprising atherapeutically effective amount of a VEGF-E polypeptide hereof inadmixture with a pharmaceutically acceptable carrier.

The invention further includes associated embodiments of VEGF-E such asmodified VEGF-E polypeptides and modified variants which have the samebiological applications as VEGF-E, and pharmaceutical compositionsincorporating same. Inhibitors of VEGF-E are also provided.

83. PRO285 and PRO286

Applicants have identified two novel cDNA clones that encode novel humanToll polypeptides, designated in the present application as PRO285(encoded by DNA40021-1154) and PRO286 (encoded by DNA42663-1154).

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising a DNA encoding a polypeptide having at least about80% sequence identity, preferably at least about 85% sequence identity,more preferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding aPRO285 polypeptide having amino acid residues 27 to 839 of FIG. 209 (SEQID NO:496); or (b) to a DNA molecule encoding a PRO286 polypeptidehaving amino acid residues 27 to 825 of FIG. 211 (SEQ ID NO:498) or (c)the complement of the DNA molecule of (a) or (b). The complementary DNAmolecule preferably remains stably bound to such encoding nucleic acidsequence under at least moderate, and optionally, under high stringencyconditions.

In a further embodiment, the isolated nucleic acid molecule comprises apolynucleotide that has at least about 90%, preferably at least about95% sequence identity with a polynucleotide encoding a polypeptidecomprising the sequence of amino acids 1 to 839 of FIG. 209 (SEQ IDNO:496); or at least about 90%, preferably at least about 95% sequenceidentity with a polynucleotide encoding a polypeptide comprising thesequence of amino acids 1 to 1041 of FIG. 211 (SEQ ID NO:498).

In a specific embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding native or variant PRO285 andPRO286 polypeptides, with or without the N-terminal signal sequence, andwith or without the transmembrane regions of the respective full-lengthsequences. In one aspect, the isolated nucleic acid comprises DNAencoding a mature, full-length native PRO285 or PRO286 polypeptidehaving amino acid residues 1 to 1049 of FIG. 209 (SEQ ID NO:496) and 1to 1041 of FIG. 211 (SEQ ID NO: 498), or is complementary to suchencoding nucleic acid sequence. In another aspect, the inventionconcerns an isolated nucleic acid molecule that comprises DNA encoding anative PRO285 or PRO286 polypeptide without an N-terminal signalsequence, or is complementary to such encoding nucleic acid sequence. Inyet another embodiment, the invention concerns nucleic acid encodingtransmembrane-domain deleted or inactivated forms of the full-lengthnative PRO285 or PRO286 proteins.

In another embodiment, the invention the isolated nucleic acid moleculecomprises the clone (DNA40021-1154) deposited on Oct. 17, 1997, underATCC number 209389; or the clone (DNA42663-1154) deposited on Oct. 17,1997, under ATCC number 209386.

In yet another embodiment, the invention provides a vector comprisingDNA encoding PRO285 and PRO286 polypeptides, or their variants. Thus,the vector may comprise any of the isolated nucleic acid moleculeshereinabove defined.

In another embodiment, the invention provides isolated PRO285 and PRO286polypeptides. In particular, the invention provides isolated nativesequence PRO285 and PRO286 polypeptides, which in one embodiment,include the amino acid sequences comprising residues 1 to 1049 and 1 to1041 of FIGS. 209 and 211 (SEQ ID NOS:496 and 498), respectively. Theinvention also provides for variants of the PRO285 and PRO286polypeptides which are encoded by any of the isolated nucleic acidmolecules hereinabove defined. Specific variants include, but are notlimited to, deletion (truncated) variants of the full-length nativesequence PRO285 and PRO286 polypeptides which lack the respectiveN-terminal signal sequences and/or have their respective transmembraneand/or cytoplasmic domains deleted or inactivated.

The invention also specifically includes antibodies with dualspecificities, e.g., bispecific antibodies binding more than one Tollpolypeptide.

In yet another embodiment, the invention concerns agonists andantagonists of the native PRO285 and PRO286 polypeptides. In aparticular embodiment, the agonist or antagonist is an anti-PRO285 oranti-PRO286 antibody.

In a further embodiment, the invention concerns screening assays toidentify agonists or antagonists of the native PRO285 and PRO286polypeptides.

In a still further embodiment, the invention concerns a compositioncomprising a PRO285 or PRO286 polypeptide, or an agonist or antagonistas hereinabove defined, in combination with a pharmaceuticallyacceptable carrier.

The invention further concerns a composition comprising an antibodyspecifically binding a PRO285 or PRO286 polypeptide, in combination witha pharmaceutically acceptable carrier.

The invention also concerns a method of treating septic shock comprisingadministering to a patient an effective amount of an antagonist of aPRO285 or PRO286 polypeptide. In a specific embodiment, the antagonistis a blocking antibody specifically binding a native PRO285 or PRO286polypeptide.

84. PRO213-1, PRO1330 and PRO1449

The present invention concerns compositions and methods for thediagnosis and treatment of neoplastic cell growth and proliferation inmammals, including humans. The present invention is based on theidentification of genes that are amplified in the genome of tumor cells.Such gene amplification is expected to be associated with theoverexpression of the gene product and contribute to tumorigenesis.Accordingly, the proteins encoded by the amplified genes are believed tobe useful targets for the diagnosis and/or treatment (includingprevention) of certain cancers, and may act as predictors of theprognosis of tumor treatment. In one embodiment, the present inventionprovides an isolated nucleic acid molecule comprising DNA encoding aPRO213-1, PRO1330 and/or PRO1449 polypeptide. In one aspect, theisolated nucleic acid comprises DNA encoding the PRO213-1, PRO1330and/or PRO1449 polypeptide having amino acid residues 1 to 295 of FIG.213 (SEQ ID NO:506), 20 to 273 of FIG. 215 (SEQ ID NO:508) and 20 to 273of FIG. 217 (SEQ ID NO:510), respectively, or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions. Theisolated nucleic acid sequence may comprise the cDNA insert of thevector designated as DNA30943-1163 (ATCC 209791) deposited on Apr. 21,1998; DNA64907-1163-1 (ATCC 203242) deposited on Sep. 9, 1998 and/orDNA64908-1163-1 (ATCC 203243) deposited on Sep. 9, 1998.

In another embodiment, the present invention comprises an isolatednucleic acid molecule having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding a PRO213-1, PRO1330and/or PRO1449 polypeptide having amino acid residues 1 to 295 of FIG.213 (SEQ ID NO:506), 20 to 273 of FIG. 215 (SEQ ID NO:508) and 20 to 273of FIG. 217 (SEQ ID NO:510), respectively; or (b) the complement of theDNA molecule of (a).

In another embodiment, the invention provides an isolated PRO213-1,PRO1330 and/or PRO1449 polypeptide. In particular, the inventionprovides isolated native sequence PRO213-1, PRO1330 and/or PRO1449polypeptide, which in one embodiment, includes an amino acid sequencecomprising residues 1 to 295 of FIG. 213 (SEQ ID NO:506), 20 to 273 ofFIG. 215 (SEQ ID NO:508) or 20 to 273 of FIG. 217 (SEQ ID NO:510),respectively. Optionally, the PRO213-1, PRO1330 and/or PRO1449polypeptide is obtained or obtainable by expressing the polypeptideencoded by the cDNA insert of the DNA30943-1163 (ATCC 209791),DNA64907-1163-1 (ATCC 203242) or DNA64908-1163-1 (ATCC 203243).

In another aspect, the invention provides an isolated PRO213-1, PRO1330,and/or PRO1449 polypeptide, comprising an amino acid sequence having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 95% sequence identityto amino acid residues 1 to 295 of FIG. 213 (SEQ ID NO:506), 20 to 273of FIG. 215 (SEQ ID NO:508) or 20 to 273 of FIG. 217 (SEQ ID NO:510),inclusive.

In yet another embodiment, the invention provides an isolated PRO213-1,PRO1330, and/or PRO1449 polypeptide, comprising the amino acid residues1 to 295 of FIG. 213 (SEQ ID NO:506), 20 to 273 of FIG. 215 (SEQ IDNO:508) or 20 to 273 of FIG. 217 (SEQ ID NO:510), or a fragment thereofsufficient to provide a binding site for an anti-PRO213-1,anti-PRO1330and/or anti-PRO1449 antibody. Preferably, the PRO213-1,PRO1330, and/or PRO1449 fragment retains a qualitative biologicalactivity of a native PRO213-1, PRO1330, and/or PRO1449 polypeptide.

In a further aspect, the invention concerns an isolated PRO213-1,PRO1330, and/or PRO1449 polypeptide, comprising an amino acid sequencescoring at least about 80% positives, preferably at least about 85%positives, more preferably at least about 90% positives, most preferablyat least about 95% positives when compared with the amino acid sequenceof residues 1 to 295 of FIG. 213 (SEQ ID NO:506), 20 to 273 of FIG. 215(SEQ ID NO:508) and 20 to 273 of FIG. 217 (SEQ ID NO:510), respectively.

In still a further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with:(a) a DNA molecule encoding a PRO213-1, PRO1330, and/or PRO1449polypeptide having the amino acid residues from 1 to 295 of FIG. 213(SEQ ID NO:506), 20 to 273 of FIG. 215 (SEQ ID NO:508) and 20 to 273 ofFIG. 217 (SEQ ID NO:510), respectively; or the complement of the DNAmolecule of (a), and if said test DNA molecule has at least about an 80%sequence identity to (a) or (b), (ii) culturing a host cell comprisingsaid test DNA molecule under conditions suitable for the expression ofsaid polypeptide, and (iii) recovering said polypeptide from the cellculture.

In one embodiment, the present invention concerns an isolated antibodywhich binds a PRO213-1, PRO1330 and/or PRO1449 polypeptide. In oneaspect, the antibody induces death of a cell overexpressing a PRO213-1,PRO1330 and/or PRO1449 polypeptide. In another aspect, the antibody is amonoclonal antibody, which preferably has nonhuman complementaritydetermining region (CDR) residues and human framework region (FR)residues. The antibody may be labeled and may be immobilized on a solidsupport. In a further aspect, the antibody is an antibody fragment, asingle-chain antibody, or an anti-idiotypic antibody.

In another embodiment, the invention concerns a composition comprisingan antibody which binds a PRO213-1, PRO1330 and/or PRO1449 polypeptidein admixture with a pharmaceutically acceptable carrier. In one aspect,the composition comprises a therapeutically effective amount of theantibody. In another aspect, the composition comprises a further activeingredient, which may, for example, be a further antibody or a cytotoxicor chemotherapeutic agent. Preferably, the composition is sterile.

In a further embodiment, the invention concerns nucleic acid encoding ananti-PRO213-1, anti-PRO1330 and/or anti-PRO1449 antibody, and vectorsand recombinant host cells comprising such nucleic acid.

The invention further concerns antagonists and agonists of a PRO213-1,PRO1330 and/or PRO1449 polypeptide that inhibit one or more of thefunctions or activities of the PRO213-1, PRO1330 and/or PRO1449polypeptide.

In a further embodiment, the invention concerns isolated nucleic acidmolecules that hybridize to the complement of the nucleic acid moleculesencoding the PRO213-1, PRO1330 and/or PRO1449 polypeptides. The nucleicacid preferably is DNA, and hybridization preferably occurs understringent conditions. Such nucleic acid molecules can act as antisensemolecules of the amplified genes identified herein, which, in turn, canfind use in the modulation of the respective amplified genes, or asantisense primers in amplification reactions. Furthermore, suchsequences can be used as part of ribozyme and/or triple helix sequencewhich, in turn, may be used in regulation of the amplified genes.

In another embodiment, the invention concerns a method for determiningthe presence of a PRO213-1, PRO1330 and/or PRO1449 polypeptidecomprising exposing a cell suspected of containing the PRO213-1, PRO1330and/or PRO1449 polypeptide to an anti-PRO213-1, PRO1330 and/or PRO1449antibody and determining binding of the antibody to the cell.

In yet another embodiment, the present invention concerns a method ofdiagnosing tumor in a mammal, comprising detecting the level ofexpression of a gene encoding a PRO213-1, PRO1330 and/or PRO1449polypeptide (a) in a test sample of tissue cells obtained from themammal, and (b) in a control sample of known normal tissue cells of thesame cell type, wherein a higher expression level in the test sampleindicates the presence of tumor in the mammal from which the test tissuecells were obtained.

In another embodiment, the present invention concerns a method ofdiagnosing tumor in a mammal, comprising (a) contacting ananti-PRO213-1, anti-PRO1330 and/or anti-PRO1449 antibody with a testsample of tissue cells obtained from the mammal, and (b) detecting theformation of a complex between the anti-PRO213-1, anti-PRO1330 and/oranti-PRO1449 antibody and the PRO213-1, PRO1330 and/or PRO1449polypeptide in the test sample. The detection may be qualitative orquantitative, and may be performed in comparison with monitoring thecomplex formation in a control sample of known normal tissue cells ofthe same cell type. A larger quantity of complexes formed in the testsample indicates the presence of tumor in the mammal from which the testtissue cells were obtained. The antibody preferably carries a detectablelabel. Complex formation can be monitored, for example, by lightmicroscopy, flow cytometry, fluorimetry, or other techniques known inthe art. The test sample is usually obtained from an individualsuspected to have neoplastic cell growth or proliferation (e.g.cancerous cells).

In another embodiment, the present invention concerns a cancerdiagnostic kit, comprising an anti-PRO213-1, anti-PRO1330 and/oranti-PRO1449 antibody and a carrier (e.g. a buffer) in suitablepackaging. The kit preferably contains instructions for using theantibody to detect the PRO213-1, PRO1330 and/or PRO1449 polypeptide.

In yet another embodiment, the invention concerns a method forinhibiting the growth of tumor cells comprising exposing a cell whichoverexpresses a PRO213-1, PRO1330 and/or PRO1449 polypeptide to aneffective amount of an agent inhibiting the expression and/or activityof the PRO213-1, PRO1330 and/or PRO1449 polypeptide. The agentpreferably is an anti-PRO213-1, anti-PRO1330 and/or anti-PRO1449antibody, a small organic and inorganic molecule, peptide,phosphopeptide, antisense or ribozyme molecule, or a triple helixmolecule. In a specific aspect, the agent, e.g. anti-PRO213-1,anti-PRO1330 and/or anti-PRO1449 antibody induces cell death. In afurther aspect, the tumor cells are further exposed to radiationtreatment and/or a cytotoxic or chemotherapeutic agent.

In a further embodiment, the invention concerns an article ofmanufacture, comprising:

-   a) a container;-   b) a label on the container; and-   c) a composition comprising an active agent contained within the    container; wherein the composition is effective for inhibiting the    growth of tumor cells, the label on the container indicates that the    composition can be used for treating conditions characterized by    overexpression of a PRO213-1, PRO1330 and/or PRO1449 polypeptide,    and the active agent in the composition is an agent inhibiting the    expression and/or activity of the PRO213-1, PRO1330 and/or PRO1449    polypeptide. In a preferred aspect, the active agent is an    anti-PRO213-1, anti-PRO1330 and/or anti-PRO1449 antibody.

In yet a further embodiment, the invention provides a method foridentifying a compound capable of inhibiting the expression and/oractivity of a PRO213-1, PRO1330 and/or PRO1449 polypeptide, comprisingcontacting a candidate compound with a PRO213-1, PRO1330 and/or PRO1449polypeptide under conditions and for a time sufficient to allow thesetwo components to interact. In a specific aspect, either the candidatecompound or the PRO213-1, PRO1330 and/or PRO1449 polypeptide isimmobilized on a solid support. In another aspect, the non-immobilizedcomponent carries a detectable label.

85. PRO298

Applicants have identified a cDNA clone that encodes a novelpolypeptide. The DNA is designated in the present application as“DNA39975-1210”, encoding a novel multi-transmembrane protein, referredto as “PRO298”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA having at least about 80%, preferably at leastabout 85%, more preferably at least about 90%, most preferably at leastabout 95% sequence identity to (a) a DNA molecule encoding PRO298,comprising the sequence of amino acids 1 to 364 of FIG. 219 (SEQ IDNO:515), or (b) the complement of the DNA molecule of (a). In oneaspect, the isolated nucleic acid comprises DNA encoding a PRO298polypeptide having amino acid residues 1 to 364 of FIG. 219 (SEQ IDNO:515), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

In a further embodiment, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least an 80% sequence identity to (a)a DNA molecule encoding the same mature polypeptide encoded by the humanprotein cDNA in ATCC Deposit No. 209783 (DNA39975-1210), or (b) thecomplement of the DNA molecule of (a).

In a still further embodiment, the invention concerns nucleic acid whichcomprises a DNA molecule encoding the same mature polypeptide encoded bythe human protein cDNA in ATCC Deposit No. 209783 (DNA39975-1210).

In another embodiment, the invention provides isolated PRO298polypeptide. In particular, the invention provides isolated nativesequence PRO298 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 364 of FIG. 219 (SEQ ID NO:515).

In another embodiment, the invention provides an expressed sequence tag(EST) designated DNA26832 comprising the nucleotide sequence of FIG. 220(SEQ ID NO:516).

86. PRO337

Applicants have identified a cDNA clone (DNA43316-1237) that encodes anovel polypeptide, designated in the present application as “PRO337”.

In one embodiment, the invention provides an isolated nucleic acidmolecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a PRO337 polypeptide comprising the sequence of aminoacids 1 to 344 of FIG. 222 (SEQ ID NO:523), or (b) the complement of theDNA molecule of (a). The sequence identity preferably is about 85%, morepreferably about 90%, most preferably about 95%. In one aspect, theisolated nucleic acid has at least about 80%, preferably at least about85%, more preferably at least about 90%, and most preferably at leastabout 95 (including 96, 97, 98 and 99%) sequence identity with apolypeptide having amino acid residues 1 to 344 of FIG. 222 (SEQ IDNO:523). Preferably, the highest degree of sequence identity occurswithin the immunoglobulin and major histocompatibility domains (aminoacids 113 to 130 of FIG. 222, SEQ ID NO:523).

In a further embodiment, the isolated nucleic acid molecule comprisesDNA encoding a neurotrimin polypeptide having amino acid residues 1 to344 of FIG. 222 (SEQ ID NO:523), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions. In anotheraspect, the invention provides a nucleic acid of the full length proteinof clone DNA43316-1237, deposited with the ATCC under accession numberATCC 209487, alternatively the coding sequence of clone DNA43316-1237,deposited under accession number ATCC 209487.

In yet another embodiment, the invention provides isolated PRO337polypeptide. In particular, the invention provides isolated nativesequence PRO337 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 344 of FIG. 222 (SEQ ID NO:523).Native PRO337 polypeptides with or without the native signal sequence(amino acids 1 to about 28 in FIG. 222 (SEQ ID NO:523), and with orwithout the initiating methionine are specifically included.Alternatively, the invention provides a PRO337 polypeptide encoded bythe nucleic acid deposited under accession number ATCC 209487.

In yet another embodiment, the invention provides an expressed sequencetag (EST) comprising the nucleotide sequences identified in FIG. 223 asDNA42301 (SEQ ID NO:524).

87. PRO403

Applicants have identified a cDNA clone (DNA55800-1263) that encodes anovel polypeptide, designated in the present application as “PRO403”.

In one embodiment, the invention provides an isolated nucleic acidmolecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a PRO403 polypeptide comprising the sequence of aminoacids 1 to 736 of FIG. 225 (SEQ ID NO:526), or (b) the complement of theDNA molecule of (a). The sequence identity preferably is about 85%, morepreferably about 90%, most preferably about 95%. In one aspect, theisolated nucleic acid has at least about 80%, preferably at least about85%, more preferably at least about 90%, and most preferably at leastabout 95% sequence identity with a polypeptide having amino acidresidues 1 to 736 of FIG. 225 (SEQ ID NO:526). Preferably, the highestdegree of sequence identity occurs within: (1) the putativeN-glycosylatation sites (amino acid residues 132, 136, 177, 237, 282,349, 505, 598 and 606; (2) Cys residues conserved with the Kell bloodgroup protein family (amino acid residues 65, 70, 88 and 96) and theputative zinc binding motif (amino acid residues 570–579).

In a further embodiment, the isolated nucleic acid molecule comprisesDNA encoding a PRO403 polypeptide having amino acid residues 1 to 736 ofFIG. 225 (SEQ ID NO:526), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. In another aspect, theinvention provides a nucleic acid of the full length protein of cloneDNA55800-1263, deposited with the ATCC under accession number ATCC209680, alternatively the coding sequence of clone DNA55800-1263,deposited under accession number ATCC 209680.

In yet another embodiment, the invention provides isolated PRO403polypeptide. In particular, the invention provides isolated nativesequence PRO403 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 736 of FIG. 225 (SEQ ID NO:526).Native PRO403 polypeptides with or the initiating methionine arespecifically included. Alternatively, the invention provides a PRO403polypeptide encoded by the nucleic acid deposited under accession numberATCC 209680.

In yet another embodiment, the invention provides an expressed sequencetag (EST) and other sequence fragments comprising the nucleotidesequences identified herein as DNA34415 (FIG. 226; SEQ ID NO:527);DNA49830 (FIG. 227; SEQ ID NO:528) and DNA49831 (FIG. 228; SEQ IDNO:529).

88. Additional Embodiments

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

In other embodiments, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PROpolypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% sequence identity, preferably atleast about 81% sequence identity, more preferably at least about 82%sequence identity, yet more preferably at least about 83% sequenceidentity, yet more preferably at least about 84% sequence identity, yetmore preferably at least about 85% sequence identity, yet morepreferably at least about 86% sequence identity, yet more preferably atleast about 87% sequence identity, yet more preferably at least about88% sequence identity, yet more preferably at least about 89% sequenceidentity, yet more preferably at least about 90% sequence identity, yetmore preferably at least about 91% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity and yet more preferablyat least about 99% sequence identity to (a) a DNA molecule encoding aPRO polypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94: % sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculecomprising the coding sequence of a full-length PRO polypeptide cDNA asdisclosed herein, the coding sequence of a PRO polypeptide lacking thesignal peptide as disclosed herein, the coding sequence of anextracellular domain of a transmembrane PRO polypeptide, with or withoutthe signal peptide, as disclosed herein or the coding sequence of anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by any of thehuman protein cDNAs deposited with the ATCC as disclosed herein, or (b)the complement of the DNA molecule of (a).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO polypeptide which iseither transmembrane domain-deleted or transmembrane domain-inactivated,or is complementary to such encoding nucleotide sequence, wherein thetransmembrane domain(s) of such polypeptide are disclosed herein.Therefore, soluble extracellular domains of the herein described PROpolypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes, for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody or as antisense oligonucleotide probes. Such nucleicacid fragments are usually at least about 20 nucleotides in length,preferably at least about 30 nucleotides in length, more preferably atleast about 40 nucleotides in length, yet more preferably at least about50 nucleotides in length, yet more preferably at least about 60nucleotides in length, yet more preferably at least about 70 nucleotidesin length, yet more preferably at least about 80 nucleotides in length,yet more preferably at least about 90 nucleotides in length, yet morepreferably at least about 100nucleotides in length, yet more preferablyat least about 110 nucleotides in length, yet more preferably at leastabout 120 nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PRO polypeptidea coding nucleotide sequence may be determined in a routine manner byaligning the PRO polypeptide-encoding nucleotide sequence with otherknown nucleotide sequences using any of a number of well known sequencealignment programs and determining which PRO polypeptide-encodingnucleotide sequence fragment(s) are novel. All of such PROpolypeptide-encoding nucleotide sequences are contemplated herein. Alsocontemplated are the PRO polypeptide fragments encoded by thesenucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides isolated PRO polypeptideencoded by any of the isolated nucleic acid sequences hereinaboveidentified.

In a certain aspect, the invention concerns an isolated PRO polypeptide,comprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by any of the human protein cDNAs deposited withthe ATCC as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence scoring at least about 80% positives,preferably at least about 81% positives, more preferably at least about82% positives, yet more preferably at least about 83% positives, yetmore preferably at least about 84% positives, yet more preferably atleast about 85% positives, yet more preferably at least about 86%positives, yet more preferably at least about 87% positives, yet morepreferably at least about 88% positives, yet more preferably at leastabout 89% positives, yet more preferably at least about 90% positives,yet more preferably at least about 91% positives, yet more preferably atleast about 92% positives, yet more preferably at least about 93%positives, yet more preferably at least about 94% positives, yet morepreferably at least about 95% positives, yet more preferably at leastabout 96% positives, yet more preferably at least about 97% positives,yet more preferably at least about 98% positives and yet more preferablyat least about 99% positives when compared with the amino acid sequenceof a PRO polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

Another aspect the invention provides an isolated PRO polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO213 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“UNQ187” and/or “DNA30943-1163”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:6) of a native sequencePRO274 cDNA, wherein SEQ ID NO:6 is a clone designated herein as“UNQ241” and/or “DNA39987-1184”.

FIG. 4 shows the amino acid sequence (SEQ ID NO:7) derived from thecoding sequence of SEQ ID NO:6 shown in FIG. 3.

FIG. 5 shows an EST nucleotide sequence designated herein as DNA17873(SEQ ID NO:8).

FIG. 6 shows an EST nucleotide sequence designated herein as DNA36157(SEQ ID NO:9).

FIG. 7 shows an EST nucleotide sequence designated herein as DNA28929(SEQ ID NO:10).

FIG. 8 shows a nucleotide sequence (SEQ ID NO:18) of a native sequencePRO300 cDNA, wherein SEQ ID NO:18 is a clone designated herein as“UNQ263” and/or “DNA40625-1189”.

FIG. 9 shows the amino acid sequence (SEQ ID NO:19) derived from thecoding sequence of SEQ ID NO:18 shown in FIG. 8.

FIG. 10 shows a nucleotide sequence (SEQ ID NO:27) of a native sequencePRO284 cDNA, wherein SEQ ID NO:27 is a clone designated herein as“UNQ247” and/or “DNA23318-1211”.

FIG. 11 shows the amino acid sequence (SEQ ID NO:28) derived from thecoding sequence of SEQ ID NO:27 shown in FIG. 10.

FIG. 12 shows an EST nucleotide sequence designated herein as DNA12982(SEQ ID NO:29).

FIG. 13 shows an EST nucleotide sequence designated herein as DNA15886(SEQ ID NO:30).

FIG. 14 shows a nucleotide sequence (SEQ ID NO:35) of a native sequencePRO296 cDNA, wherein SEQ ID NO:35 is a clone designated herein as“UNQ260” and/or “DNA39979-1213”.

FIG. 15 shows the amino acid sequence (SEQ ID NO:36) derived from thecoding sequence of SEQ ID NO:35 shown in FIG. 14.

FIG. 16 shows an EST nucleotide sequence designated herein as DNA3020(SEQ ID NO:37).

FIG. 17 shows an EST nucleotide sequence designated herein as DNA21971(SEQ ID NO:38).

FIG. 18 shows an EST nucleotide sequence designated herein as DNA29037(SEQ ID NO:39).

FIG. 19 shows a nucleotide sequence (SEQ ID NO:44) of a native sequencePRO329 cDNA, wherein SEQ ID NO:44 is a clone designated herein as“UNQ291” and/or “DNA40594-1233”.

FIG. 20 shows the amino acid sequence (SEQ ID NO:45) derived from thecoding sequence of SEQ ID NO:44 shown in FIG. 19.

FIG. 21 shows a nucleotide sequence (SEQ ID NO:51) of a native sequencePRO362 cDNA, wherein SEQ ID NO:51 is a clone designated herein as“UNQ317” and/or “DNA4541-1251”.

FIG. 22 shows the amino acid sequence (SEQ ID NO:52) derived from thecoding sequence of SEQ ID NO:51 shown in FIG. 21.

FIG. 23 shows a nucleotide sequence (SEQ ID NO:58) of a native sequencePRO363 cDNA, wherein SEQ ID NO:58 is a clone designated herein as“UNQ318” and/or “DNA45419-1252”.

FIG. 24 shows the amino acid sequence (SEQ ID NO:59) derived from thecoding sequence of SEQ ID NO:58 shown in FIG. 23.

FIG. 25 shows a nucleotide sequence (SEQ ID NO:63) of a native sequencePRO868 cDNA, wherein SEQ ID NO:63 is a clone designated herein as“UNQ437” and/or “DNA52594-1270”.

FIG. 26 shows the amino acid sequence (SEQ ID NO:64) derived from thecoding sequence of SEQ ID NO:63 shown in FIG. 25.

FIG. 27 shows a nucleotide sequence (SEQ ID NO:68) of a native sequencePRO382 cDNA, wherein SEQ ID NO:68 is a clone designated herein as“UNQ323” and/or “DNA45234-1277”.

FIG. 28 shows the amino acid sequence (SEQ ID NO:69) derived from thecoding sequence of SEQ ID NO:68 shown in FIG. 27.

FIG. 29 shows a nucleotide sequence (SEQ ID NO:73) of a native sequencePRO545 cDNA, wherein SEQ ID NO:73 is a clone designated herein as“UNQ346” and/or “DNA49624-1279”.

FIG. 30 shows the amino acid sequence (SEQ ID NO:74) derived from thecoding sequence of SEQ ID NO:73 shown in FIG. 29.

FIG. 31 shows an EST nucleotide sequence designated herein as DNA13217(SEQ ID NO:75).

FIG. 32 shows a nucleotide sequence (SEQ ID NO:84) of a native sequencePRO617 cDNA, wherein SEQ ID NO:84 is a clone designated herein as“UNQ353” and/or “DNA48309-1280”.

FIG. 33 shows the amino acid sequence (SEQ ID NO:85) derived from thecoding sequence of SEQ ID NO:84 shown in FIG. 32.

FIG. 34 shows a nucleotide sequence (SEQ ID NO:89) of a native sequencePRO700 cDNA, wherein SEQ ID NO:89 is a clone designated herein as“UNQ364” and/or “DNA46776-1284”.

FIG. 35 shows the amino acid sequence (SEQ ID NO:90) derived from thecoding sequence of SEQ ID NO:89 shown in FIG. 34.

FIG. 36 shows a nucleotide sequence (SEQ ID NO:96) of a native sequencePRO702 cDNA, wherein SEQ ID NO:96 is a clone designated herein as“UNQ366” and/or “DNA50980-1286”.

FIG. 37 shows the amino acid sequence (SEQ ID NO:97) derived from thecoding sequence of SEQ ID NO:96 shown in FIG. 36.

FIG. 38 shows a nucleotide sequence (SEQ ID NO:101) of a native sequencePRO703 cDNA, wherein SEQ ID NO:101 is a clone designated herein as“UNQ367” and/or “DNA50913-1287”.

FIG. 39 shows the amino acid sequence (SEQ ID NO:102) derived from thecoding sequence of SEQ ID NO:101 shown in FIG. 38.

FIG. 40 shows a nucleotide sequence (SEQ ID NO:108) of a native sequencePRO705 cDNA, wherein SEQ ID NO:108 is a clone designated herein as“UNQ369” and/or “DNA50914-1289”.

FIG. 41 shows the amino acid sequence (SEQ ID NO:109) derived from thecoding sequence of SEQ ID NO:108 shown in FIG. 40.

FIGS. 42A–B show a nucleotide sequence (SEQ ID NO:113) of a nativesequence PRO708 cDNA, wherein SEQ ID NO:113 is a clone designated hereinas “UNQ372” and/or “DNA48296-1292”.

FIG. 43 shows the amino acid sequence (SEQ ID NO:114) derived from thecoding sequence of SEQ ID NO:113 shown in FIGS. 42A–B.

FIG. 44 shows a nucleotide sequence (SEQ ID NO:118) of a native sequencePRO320 cDNA, wherein SEQ ID NO:118 is a clone designated herein as“UNQ281” and/or “DNA32284-1307”.

FIG. 45 shows the amino acid sequence (SEQ ID NO:119) derived from thecoding sequence of SEQ ID NO:118 shown in FIG. 44.

FIG. 46 shows a nucleotide sequence (SEQ ID NO:123) of a native sequencePRO324 cDNA, wherein SEQ ID NO:123 is a clone designated herein as“UNQ285” and/or “DNA36343-1310”.

FIG. 47 shows the amino acid sequence (SEQ ID NO:124) derived from thecoding sequence of SEQ ID NO:123 shown in FIG. 46.

FIG. 48 shows a nucleotide sequence (SEQ ID NO:131) of a native sequencePRO351 cDNA, wherein SEQ ID NO:131 is a clone designated herein as“UNQ308” and/or “DNA40571-1315”.

FIG. 49 shows the amino acid sequence (SEQ ID NO:132) derived from thecoding sequence of SEQ ID NO:131 shown in FIG. 48.

FIG. 50 shows a nucleotide sequence (SEQ ID NO:136) of a native sequencePRO352 cDNA, wherein SEQ ID NO:136 is a clone designated herein as“UNQ309” and/or “DNA41386-1316”.

FIG. 51 shows the amino acid sequence (SEQ ID NO:137) derived from thecoding sequence of SEQ ID NO:136 shown in FIG. 50.

FIG. 52 shows a nucleotide sequence (SEQ ID NO:144) of a native sequencePRO381 cDNA, wherein SEQ ID NO:144 is a clone designated herein as“UNQ322” and/or “DNA44194-1317”.

FIG. 53 shows the amino acid sequence (SEQ ID NO:145) derived from thecoding sequence of SEQ ID NO:144 shown in FIG. 52.

FIG. 54 shows a nucleotide sequence (SEQ ID NO:149) of a native sequencePRO386 cDNA, wherein SEQ ID NO:149 is a clone designated herein as“UNQ326” and/or “DNA45415-1318”.

FIG. 55 shows the amino acid sequence (SEQ ID NO:150) derived from thecoding sequence of SEQ ID NO:149 shown in FIG. 54.

FIG. 56 shows an EST nucleotide sequence designated herein as DNA23350(SEQ ID NO:151).

FIG. 57 shows an EST nucleotide sequence designated herein as DNA23536(SEQ ID NO:152).

FIG. 58 shows a nucleotide sequence (SEQ ID NO:156) of a native sequencePRO540 cDNA, wherein SEQ ID NO:156 is a clone designated herein as“UNQ341” and/or “DNA44189-1322”.

FIG. 59 shows the amino acid sequence (SEQ ID NO:157) derived from thecoding sequence of SEQ ID NO:156 shown in FIG. 58.

FIG. 60 shows a nucleotide sequence (SEQ ID NO:161) of a native sequencePRO615 cDNA, wherein SEQ ID NO:161 is a clone designated herein as“UNQ352” and/or “DNA48304-1323”.

FIG. 61 shows the amino acid sequence (SEQ ID NO:162) derived from thecoding sequence of SEQ ID NO:161 shown in FIG. 60.

FIG. 62 shows a nucleotide sequence (SEQ ID NO:168) of a native sequencePRO618 cDNA, wherein SEQ ID NO:168 is a clone designated herein as“UNQ354” and/or “DNA49152-1324”.

FIG. 63 shows the amino acid sequence (SEQ ID NO:169) derived from thecoding sequence of SEQ ID NO:168 shown in FIG. 62.

FIG. 64 shows an EST nucleotide sequence designated herein as DNA35597(SEQ ID NO:170).

FIG. 65 shows a nucleotide sequence (SEQ ID NO:177) of a native sequencePRO719 cDNA, wherein SEQ ID NO:177 is a clone designated herein as“UNQ387” and/or “DNA49646-1327”.

FIG. 66 shows the amino acid sequence (SEQ ID NO:178) derived from thecoding sequence of SEQ ID NO:177 shown in FIG. 65.

FIG. 67 shows a nucleotide sequence (SEQ ID NO:182) of a native sequencePRO724 cDNA, wherein SEQ ID NO:182 is a clone designated herein as“UNQ389” and/or “DNA49631-1328”.

FIG. 68 shows the amino acid sequence (SEQ ID NO:183) derived from thecoding sequence of SEQ ID NO:182 shown in FIG. 67.

FIG. 69 shows a nucleotide sequence (SEQ ID NO:189) of a native sequencePRO772 cDNA, wherein SEQ ID NO:189 is a clone designated herein as“UNQ410” and/or “DNA49645-1347”.

FIG. 70 shows the amino acid sequence (SEQ ID NO:190) derived from thecoding sequence of SEQ ID NO:189 shown in FIG. 69.

FIG. 71 shows an EST nucleotide sequence designated herein as DNA43509(SEQ ID NO:191).

FIG. 72 shows a nucleotide sequence (SEQ ID NO:195) of a native sequencePRO852 cDNA, wherein SEQ ID NO:195 is a clone designated herein as“UNQ418” and/or “DNA45493-1349”.

FIG. 73 shows the amino acid sequence (SEQ ID NO:196) derived from thecoding sequence of SEQ ID NO:195 shown in FIG. 72.

FIG. 74 shows a nucleotide sequence (SEQ ID NO:205) of a native sequencePRO853 cDNA, wherein SEQ ID NO:205 is a clone designated herein as“UNQ419” and/or “DNA48227-1350”.

FIG. 75 shows the amino acid sequence (SEQ ID NO:206) derived from thecoding sequence of SEQ ID NO:205 shown in FIG. 74.

FIG. 76 shows a nucleotide sequence (SEQ ID NO:210) of a native sequencePRO860 cDNA, wherein SEQ ID NO:210 is a clone designated herein as“UNQ421” and/or “DNA41404-1352”.

FIG. 77 shows the amino acid sequence (SEQ ID NO:211) derived from thecoding sequence of SEQ ID NO:210 shown in FIG. 76.

FIG. 78 shows a nucleotide sequence (SEQ ID NO:215) of a native sequencePRO846 cDNA, wherein SEQ ID NO:215 is a clone designated herein as“UNQ422” and/or “DNA44196-1353”.

FIG. 79 shows the amino acid sequence (SEQ ID NO:216) derived from thecoding sequence of SEQ ID NO:215 shown in FIG. 78.

FIG. 80 shows a nucleotide sequence (SEQ ID NO:220) of a native sequencePRO862 cDNA, wherein SEQ ID NO:220 is a clone designated herein as“UNQ424” and/or “DNA52187-1354”.

FIG. 81 shows the amino acid sequence (SEQ ID NO:221) derived from thecoding sequence of SEQ ID NO:220 shown in FIG. 80.

FIG. 82 shows a nucleotide sequence (SEQ ID NO:225) of a native sequencePRO864 cDNA, wherein SEQ ID NO:225 is a clone designated herein as“UNQ426” and/or “DNA48328-1355”.

FIG. 83 shows the amino acid sequence (SEQ ID NO:226) derived from thecoding sequence of SEQ ID NO:225 shown in FIG. 82.

FIG. 84 shows a nucleotide sequence (SEQ ID NO:230) of a native sequencePRO792 cDNA, wherein SEQ ID NO:230 is a clone designated herein as“UNQ431” and/or “DNA56352-1358”.

FIG. 85 shows the amino acid sequence (SEQ ID NO:231) derived from thecoding sequence of SEQ ID NO:230 shown in FIG. 84.

FIG. 86 shows a nucleotide sequence (SEQ ID NO:235) of a native sequencePRO866 cDNA, wherein SEQ ID NO:235 is a clone designated herein as“UNQ435” and/or “DNA53971-1359”.

FIG. 87 shows the amino acid sequence (SEQ ID NO:236) derived from thecoding sequence of SEQ ID NO:235 shown in FIG. 86.

FIG. 88 shows a nucleotide sequence (SEQ ID NO:244) of a native sequencePRO871 cDNA, wherein SEQ ID NO:244 is a clone designated herein as“UNQ438” and/or “DNA50919-1361”.

FIG. 89 shows the amino acid sequence (SEQ ID NO:245) derived from thecoding sequence of SEQ ID NO:244 shown in FIG. 88.

FIG. 90 shows a nucleotide sequence (SEQ ID NO:253) of a native sequencePRO873 cDNA, wherein SEQ ID NO:253 is a clone designated herein as“UNQ440” and/or “DNA44179-1362”.

FIG. 91 shows the amino acid sequence (SEQ ID NO:254) derived from thecoding sequence of SEQ ID NO:253 shown in FIG. 90.

FIG. 92 shows a nucleotide sequence (SEQ ID NO:258) of a native sequencePRO940 cDNA, wherein SEQ ID NO:258 is a clone designated herein as“UNQ477” and/or “DNA54002-1367”.

FIG. 93 shows the amino acid sequence (SEQ ID NO:259) derived from thecoding sequence of SEQ ID NO:258 shown in FIG. 92.

FIG. 94 shows a nucleotide sequence (SEQ ID NO:263) of a native sequencePRO941 cDNA, wherein SEQ ID NO:263 is a clone designated herein as“UNQ478” and/or “DNA53906-1368”.

FIG. 95 shows the amino acid sequence (SEQ ID NO:264) derived from thecoding sequence of SEQ ID NO:263 shown in FIG. 94.

FIG. 96 shows an EST nucleotide sequence designated herein as DNA6415(SEQ ID NO:265).

FIG. 97 shows a nucleotide sequence (SEQ ID NO:269) of a native sequencePRO944 cDNA, wherein SEQ ID NO:269 is a clone designated herein as“UNQ481” and/or “DNA52185-1370”.

FIG. 98 shows the amino acid sequence (SEQ ID NO:270) derived from thecoding sequence of SEQ ID NO:269 shown in FIG. 97.

FIG. 99 shows an EST nucleotide sequence designated herein as DNA14007(SEQ ID NO:271).

FIG. 100 shows an EST nucleotide sequence designated herein as DNA12773(SEQ ID NO:272).

FIG. 101 shows an EST nucleotide sequence designated herein as DNA12746(SEQ ID NO:273).

FIG. 102 shows an EST nucleotide sequence designated herein as DNA12834(SEQ ID NO:274).

FIG. 103 shows an EST nucleotide sequence designated herein as DNA12846(SEQ ID NO:275).

FIG. 104 shows an EST nucleotide sequence designated herein as DNA13104(SEQ ID NO:276).

FIG. 105 shows an EST nucleotide sequence designated herein as DNA13259(SEQ ID NO:277).

FIG. 106 shows an EST nucleotide sequence designated herein as DNA13959(SEQ ID NO:278).

FIG. 107 shows an EST nucleotide sequence designated herein as DNA13961(SEQ ID NO:279).

FIG. 108 shows a nucleotide sequence (SEQ ID NO:283) of a nativesequence PRO983 cDNA, wherein SEQ ID NO:283 is a clone designated hereinas “UNQ484” and/or “DNA53977-1371”.

FIG. 109 shows the amino acid sequence (SEQ ID NO:284) derived from thecoding sequence of SEQ ID NO:283 shown in FIG. 108.

FIG. 110 shows an EST nucleotide sequence designated herein as DNA17130(SEQ ID NO:285).

FIG. 111 shows an EST nucleotide sequence designated herein as DNA23466(SEQ ID NO:286).

FIG. 112 shows an EST nucleotide sequence designated herein as DNA26818(SEQ ID NO:287).

FIG. 113 shows an EST nucleotide sequence designated herein as DNA37618(SEQ ID NO:288).

FIG. 114 shows an EST nucleotide sequence designated herein as DNA41732(SEQ ID NO:289).

FIG. 115 shows an EST nucleotide sequence designated herein as DNA45980(SEQ ID NO:290).

FIG. 116 shows an EST nucleotide sequence designated herein as DNA46372(SEQ ID NO:291).

FIG. 117 shows a nucleotide sequence (SEQ ID NO:295) of a nativesequence PRO1057 cDNA, wherein SEQ ID NO:295 is a clone designatedherein as “UNQ522” and/or “DNA57253-1382”.

FIG. 118 shows the amino acid sequence (SEQ ID NO:296) derived from thecoding sequence of SEQ ID NO:295 shown in FIG. 117.

FIG. 119 shows a nucleotide sequence (SEQ ID NO:300) of a nativesequence PRO1071 cDNA, wherein SEQ ID NO:300 is a clone designatedherein as “UNQ528” and/or “DNA58847-1383”.

FIG. 120 shows the amino acid sequence (SEQ ID NO:301) derived from thecoding sequence of SEQ ID NO:300 shown in FIG. 119.

FIG. 121 shows a nucleotide sequence (SEQ ID NO:302) of a nativesequence PRO1072 cDNA, wherein SEQ ID NO:302 is a clone designatedherein as “UNQ529” and/or “DNA58747-1384”.

FIG. 122 shows the amino acid sequence (SEQ ID NO:303) derived from thecoding sequence of SEQ ID NO:302 shown in FIG. 121.

FIG. 123 shows an EST nucleotide sequence designated herein as DNA40210(SEQ ID NO:304).

FIG. 124 shows a nucleotide sequence (SEQ ID NO:308) of a nativesequence PRO1075 cDNA, wherein SEQ ID NO:308 is a clone designatedherein as “UNQ532” and/or “DNA57689-1385”.

FIG. 125 shows the amino acid sequence (SEQ ID NO:309) derived from thecoding sequence of SEQ ID NO:308 shown in FIG. 124.

FIG. 126 shows an EST nucleotide sequence designated herein as DNA13059(SEQ ID NO:310).

FIG. 127 shows an EST nucleotide sequence designated herein as DNA19463(SEQ ID NO:310).

FIG. 128 shows a nucleotide sequence (SEQ ID NO:321) of a nativesequence PRO181 cDNA, wherein SEQ ID NO:321 is a clone designated hereinas “UNQ155” and/or “DNA23330-1390”.

FIG. 129 shows the amino acid sequence (SEQ ID NO:322) derived from thecoding sequence of SEQ ID NO:321 shown in FIG. 128.

FIG. 130 shows an EST nucleotide sequence designated herein as DNA13242(SEQ ID NO:323).

FIG. 131 shows a nucleotide sequence (SEQ ID NO:329) of a nativesequence PRO195 cDNA, wherein SEQ ID NO:329 is a clone designated hereinas “UNQ169” and/or “DNA26847-1395”.

FIG. 132 shows the amino acid sequence (SEQ ID NO:330) derived from thecoding sequence of SEQ ID NO:329 shown in FIG. 131.

FIG. 133 shows an EST nucleotide sequence designated herein as DNA15062(SEQ ID NO:331).

FIG. 134 shows an EST nucleotide sequence designated herein as DNA13199(SEQ ID NO:332).

FIG. 135 shows a nucleotide sequence (SEQ ID NO:336) of a nativesequence PRO865 cDNA, wherein SEQ ID NO:336 is a clone designated hereinas “UNQ434” and/or “DNA53974-1401”.

FIG. 136 shows the amino acid sequence (SEQ ID NO:337) derived from thecoding sequence of SEQ ID NO:336 shown in FIG. 135.

FIG. 137 shows an EST nucleotide sequence designated herein as DNA37642(SEQ ID NO:338).

FIG. 138 shows a nucleotide sequence (SEQ ID NO:345) of a nativesequence PRO827 cDNA, wherein SEQ ID NO:345 is a clone designated hereinas “UNQ468” and/or “DNA57039-1402”.

FIG. 139 shows the amino acid sequence (SEQ ID NO:346) derived from thecoding sequence of SEQ ID NO:345 shown in FIG. 138.

FIG. 140 shows an EST nucleotide sequence designated herein as DNA47751(SEQ ID NO:347).

FIG. 141 shows a nucleotide sequence (SEQ ID NO:351) of a nativesequence PRO114 cDNA, wherein SEQ ID NO:351 is a clone designated hereinas “UNQ557” and/or “DNA57033-1403”.

FIG. 142 shows the amino acid sequence (SEQ ID NO:352) derived from thecoding sequence of SEQ ID NO:351 shown in FIG. 141.

FIG. 143 shows an EST nucleotide sequence designated herein as DNA48466(SEQ ID NO:353).

FIG. 144 shows a nucleotide sequence (SEQ ID NO:357) of a nativesequence PRO237 cDNA, wherein SEQ ID NO:357 is a clone designated hereinas “UNQ211 and/or “DNA34353-1428”.

FIG. 145 shows the amino acid sequence (SEQ ID NO:358) derived from thecoding sequence of SEQ ID NO:357 shown in FIG. 144.

FIG. 146 shows a nucleotide sequence (SEQ ID NO:362) of a nativesequence PRO541 cDNA, wherein SEQ ID NO:362 is a clone designated hereinas “UNQ342” and/or “DNA45417-1432”.

FIG. 147 shows the amino acid sequence (SEQ ID NO:363) derived from thecoding sequence of SEQ ID NO:362 shown in FIG. 146.

FIG. 148 shows a nucleotide sequence (SEQ ID NO:369) of a nativesequence PRO273 cDNA, wherein SEQ ID NO:369 is a clone designated hereinas “UNQ240” and/or “DNA39523-1192”.

FIG. 149 shows the amino acid sequence (SEQ ID NO:370) derived from thecoding sequence of SEQ ID NO:369 shown in FIG. 148.

FIG. 150 shows a nucleotide sequence (SEQ ID NO:374) of a nativesequence PRO701 cDNA, wherein SEQ ID NO:374 is a clone designated hereinas “UNQ365” and/or “DNA44205-1285”.

FIG. 151 shows the amino acid sequence (SEQ ID NO:375) derived from thecoding sequence of SEQ ID NO:374 shown in FIG. 150.

FIG. 152 shows a nucleotide sequence (SEQ ID NO:379) of a nativesequence PRO704 cDNA, wherein SEQ ID NO:379 is a clone designated hereinas “UNQ368” and/or “DNA50911-1288”.

FIG. 153 shows the amino acid sequence (SEQ ID NO:380) derived from thecoding sequence of SEQ ID NO:379 shown in FIG. 152.

FIG. 154 shows a nucleotide sequence (SEQ ID NO:384) of a nativesequence PRO706 cDNA, wherein SEQ ID NO:384 is a clone designated hereinas “UNQ370” and/or “DNA48329-1290”.

FIG. 155 shows the amino acid sequence (SEQ ID NO:385) derived from thecoding sequence of SEQ ID NO:384 shown in FIG. 154.

FIG. 156 shows a nucleotide sequence (SEQ ID NO:389) of a nativesequence PRO707 cDNA, wherein SEQ ID NO:389 is a clone designated hereinas “UNQ371” and/or “DNA48306-1291”.

FIG. 157 shows the amino acid sequence (SEQ ID NO:390) derived from thecoding sequence of SEQ ID NO:389 shown in FIG. 156.

FIG. 158 shows a nucleotide sequence (SEQ ID NO:394) of a nativesequence PRO322 cDNA, wherein SEQ ID NO:394 is a clone designated hereinas “UNQ283” and/or “DNA48336-1309”.

FIG. 159 shows the amino acid sequence (SEQ ID NO:395) derived from thecoding sequence of SEQ ID NO:394 shown in FIG. 158.

FIG. 160 shows a nucleotide sequence (SEQ ID NO:399) of a nativesequence PRO526 cDNA, wherein SEQ ID NO:399 is a clone designated hereinas “UNQ330” and/or “DNA44184-1319”.

FIG. 161 shows the amino acid sequence (SEQ ID NO:400) derived from thecoding sequence of SEQ ID NO:399 shown in FIG. 160.

FIG. 162 shows a nucleotide sequence (SEQ ID NO:404) of a nativesequence PRO531 cDNA, wherein SEQ ID NO:404 is a clone designated hereinas “UNQ332” and/or “DNA48314-1320”.

FIG. 163 shows the amino acid sequence (SEQ ID NO:405) derived from thecoding sequence of SEQ ID NO:404 shown in FIG. 162.

FIG. 164 shows a nucleotide sequence (SEQ ID NO:409) of a nativesequence PRO534 cDNA, wherein SEQ ID NO:409 is a clone designated hereinas “UNQ335” and/or “DNA48333-1321”.

FIG. 165 shows the amino acid sequence (SEQ ID NO:410) derived from thecoding sequence of SEQ ID NO:409 shown in FIG. 164.

FIG. 166 shows a nucleotide sequence (SEQ ID NO:414) of a nativesequence PRO697 cDNA, wherein SEQ ID NO:414 is a clone designated hereinas “UNQ361” and/or “DNA50920-1325”.

FIG. 167 shows the amino acid sequence (SEQ ID NO:415) derived from thecoding sequence of SEQ ID NO:414 shown in FIG. 166.

FIG. 168 shows a nucleotide sequence (SEQ ID NO:419) of a nativesequence PRO717 cDNA, wherein SEQ ID NO:419 is a clone designated hereinas “UNQ385” and/or “DNA50988-1326”.

FIG. 169 shows the amino acid sequence (SEQ ID NO:420) derived from thecoding sequence of SEQ ID NO:419 shown in FIG. 168.

FIG. 170 shows a nucleotide sequence (SEQ ID NO:424) of a nativesequence PRO731 cDNA, wherein SEQ ID NO:424 is a clone designated hereinas “UNQ395” and/or “DNA48331-1329”.

FIG. 171 shows the amino acid sequence (SEQ ID NO:425) derived from thecoding sequence of SEQ ID NO:424 shown in FIG. 170.

FIG. 172 shows a nucleotide sequence (SEQ ID NO:429) of a nativesequence PRO218 cDNA, wherein SEQ ID NO:429 is a clone designated hereinas “UNQ192” and/or “DNA30867-1335”.

FIG. 173 shows the amino acid sequence (SEQ ID NO:430) derived from thecoding sequence of SEQ ID NO:429 shown in FIG. 172.

FIG. 174 shows an EST nucleotide sequence designated herein as DNA14472(SEQ ID NO:431).

FIG. 175 shows an EST nucleotide sequence designated herein as DNA15846(SEQ ID NO:432).

FIG. 176 shows a nucleotide sequence (SEQ ID NO:436) of a nativesequence PRO768 cDNA, wherein SEQ ID NO:436 is a clone designated hereinas “UNQ406” and/or “DNA55737-1345”.

FIG. 177 shows the amino acid sequence (SEQ ID NO:437) derived from thecoding sequence of SEQ ID NO:436 shown in FIG. 176.

FIG. 178 shows a nucleotide sequence (SEQ ID NO:441) of a nativesequence PRO771 cDNA, wherein SEQ ID NO:441 is a clone designated hereinas “UNQ409” and/or “DNA49829-1346”.

FIG. 179 shows the amino acid sequence (SEQ ID NO:442) derived from thecoding sequence of SEQ ID NO:441 shown in FIG. 178.

FIG. 180 shows a nucleotide sequence (SEQ ID NO:446) of a nativesequence PRO733 cDNA, wherein SEQ ID NO:446 is a clone designated hereinas “UNQ411” and/or “DNA52196-1348”.

FIG. 181 shows the amino acid sequence (SEQ ID NO:447) derived from thecoding sequence of SEQ ID NO:446 shown in FIG. 180.

FIG. 182 shows a nucleotide sequence (SEQ ID NO:451) of a nativesequence PRO162 cDNA, wherein SEQ ID NO:451 is a clone designated hereinas “UNQ429” and/or “DNA56965-1356”.

FIG. 183 shows the amino acid sequence (SEQ ID NO:452) derived from thecoding sequence of SEQ ID NO:451 shown in FIG. 182.

FIG. 184 shows a nucleotide sequence (SEQ ID NO:453) of a nativesequence PRO788 cDNA, wherein SEQ ID NO:453 is a clone designated hereinas “UNQ430” and/or “DNA56405-1357”.

FIG. 185 shows the amino acid sequence (SEQ ID NO:454) derived from thecoding sequence of SEQ ID NO:453 shown in FIG. 184.

FIG. 186 shows a nucleotide sequence (SEQ ID NO:455) of a nativesequence PRO1008 cDNA, wherein SEQ ID NO:455 is a clone designatedherein as “UNQ492” and/or “DNA57530-1375”.

FIG. 187 shows the amino acid sequence (SEQ ID NO:456) derived from thecoding sequence of SEQ ID NO:455 shown in FIG. 186.

FIG. 188 shows an EST nucleotide sequence designated herein as DNA16508(SEQ ID NO:457).

FIG. 189 shows a nucleotide sequence (SEQ ID NO:458) of a nativesequence PRO1012 cDNA, wherein SEQ ID NO:458 is a clone designatedherein as “UNQ495” and/or “DNA56439-1376”.

FIG. 190 shows the amino acid sequence (SEQ ID NO:459) derived from thecoding sequence of SEQ ID NO:458 shown in FIG. 189.

FIG. 191 shows a nucleotide sequence (SEQ ID NO:463) of a nativesequence PRO1014 cDNA, wherein SEQ ID NO:463 is a clone designatedherein as “UNQ497” and/or “DNA56409-1377”.

FIG. 192 shows the amino acid sequence (SEQ ID NO:464) derived from thecoding sequence of SEQ ID NO:463 shown in FIG. 191.

FIG. 193 shows a nucleotide sequence (SEQ ID NO:465) of a nativesequence PRO1017 cDNA, wherein SEQ ID NO:465 is a clone designatedherein as “UNQ500” and/or “DNA56112-1379”.

FIG. 194 shows the amino acid sequence (SEQ ID NO:466) derived from thecoding sequence of SEQ ID NO:465 shown in FIG. 193.

FIG. 195 shows a nucleotide sequence (SEQ ID NO:467) of a nativesequence PRO474 cDNA, wherein SEQ ID NO:467 is a clone designated hereinas “UNQ502” and/or “DNA56045-1380”.

FIG. 196 shows the amino acid sequence (SEQ ID NO:468) derived from thecoding sequence of SEQ ID NO:467 shown in FIG. 195.

FIG. 197 shows a nucleotide sequence (SEQ ID NO:469) of a nativesequence PRO1031 cDNA, wherein SEQ ID NO:469 is a clone designatedherein as “UNQ516” and/or “DNA59294-1381”.

FIG. 198 shows the amino acid sequence (SEQ ID NO:470) derived from thecoding sequence of SEQ ID NO:469 shown in FIG. 197.

FIG. 199 shows a nucleotide sequence (SEQ ID NO:471) of a nativesequence PRO938 cDNA, wherein SEQ ID NO:471 is a clone designated hereinas “UNQ475” and/or “DNA56433-1406”.

FIG. 200 shows the amino acid sequence (SEQ ID NO:472) derived from thecoding sequence of SEQ ID NO:471 shown in FIG. 199.

FIG. 201 shows a nucleotide sequence (SEQ ID NO:476) of a nativesequence PRO1082 cDNA, wherein SEQ ID NO:476 is a clone designatedherein as “UNQ539” and/or “DNA53912-1457”.

FIG. 202 shows the amino acid sequence (SEQ ID NO:477) derived from thecoding sequence of SEQ ID NO:476 shown in FIG. 201.

FIG. 203 shows a nucleotide sequence (SEQ ID NO:482) of a nativesequence PRO1083 cDNA, wherein SEQ ID NO:482 is a clone designatedherein as “UNQ540” and/or “DNA50921-1458”.

FIG. 204 shows the amino acid sequence (SEQ ID NO:483) derived from thecoding sequence of SEQ ID NO:482 shown in FIG. 203.

FIG. 205 shows an EST nucleotide sequence designated herein as DNA24256(SEQ ID NO:484).

FIG. 206 shows a nucleotide sequence (SEQ ID NO:487) of a nativesequence PRO200 cDNA, wherein SEQ ID NO:487 is a clone designated hereinas “UNQ174” and/or “DNA29101-1122”.

FIG. 207 shows the amino acid sequence (SEQ ID NO:488) derived from thecoding sequence of SEQ ID NO:487 shown in FIG. 206.

FIG. 208 shows a nucleotide sequence (SEQ ID NO:495) of a nativesequence PRO285 cDNA, wherein SEQ ID NO:495 is a clone designated hereinas “DNA40021-1154”.

FIG. 209 shows the amino acid sequence (SEQ ID NO:496) derived from thecoding sequence of SEQ ID NO:495 shown in FIG. 208.

FIG. 210 shows a nucleotide sequence (SEQ ID NO:497) of a nativesequence PRO286 cDNA, wherein SEQ ID NO:497 is a clone designated hereinas “DNA42663-1154”.

FIG. 211 shows the amino acid sequence (SEQ ID NO:498) derived from thecoding sequence of SEQ ID NO:497 shown in FIG. 210.

FIG. 212 shows a nucleotide sequence (SEQ ID NO:505) of a nativesequence PRO213-1 cDNA, wherein SEQ ID NO:505 is a clone designatedherein as “DNA30943-1-1163-1”.

FIG. 213 shows the amino acid sequence (SEQ ID NO:506) derived from thecoding sequence of SEQ ID NO:505 shown in FIG. 212.

FIG. 214 shows a nucleotide sequence (SEQ ID NO:507) of a nativesequence PRO1330 cDNA, wherein SEQ ID NO:507 is a clone designatedherein as “DNA64907-1163-1”.

FIG. 215 shows the amino acid sequence (SEQ ID NO:508) derived from thecoding sequence of SEQ ID NO:507 shown in FIG. 214.

FIG. 216 shows a nucleotide sequence (SEQ ID NO:509) of a nativesequence PRO1449 cDNA, wherein SEQ ID NO:509 is a clone designatedherein as “DNA64908-1163-1”.

FIG. 217 shows the amino acid sequence (SEQ ID NO:510) derived from thecoding sequence of SEQ ID NO:509 shown in FIG. 216.

FIG. 218 shows a nucleotide sequence (SEQ ID NO:514) of a nativesequence PRO298 cDNA, wherein SEQ ID NO:514 is a clone designated hereinas “UNQ261” and/or “DNA39975-1210”.

FIG. 219 shows the amino acid sequence (SEQ ID NO:515) derived from thecoding sequence of SEQ ID NO:514 shown in FIG. 218.

FIG. 220 shows an EST nucleotide sequence designated herein as DNA26832(SEQ ID NO:516).

FIG. 221 shows a nucleotide sequence (SEQ ID NO:522) of a nativesequence PRO337 cDNA, wherein SEQ ID NO:522 is a clone designated hereinas “DNA43316-1237”.

FIG. 222 shows the amino acid sequence (SEQ ID NO:523) derived from thecoding sequence of SEQ ID NO:522 shown in FIG. 221.

FIG. 223 shows an EST nucleotide sequence designated herein as DNA42301(SEQ ID NO:524).

FIG. 224 shows a nucleotide sequence (SEQ ID NO:525) of a nativesequence PRO403 cDNA, wherein SEQ ID NO:525 is a clone designated hereinas “DNA55800-1263”.

FIG. 225 shows the amino acid sequence (SEQ ID NO:526) derived from thecoding sequence of SEQ ID NO:525 shown in FIG. 224.

FIG. 226 shows an EST nucleotide sequence designated herein as DNA34415(SEQ ID NO:527).

FIG. 227 shows an EST nucleotide sequence designated herein as DNA49830(SEQ ID NO:528).

FIG. 228 shows an EST nucleotide sequence designated herein as DNA49831(SEQ ID NO:529).

FIG. 229 shows a nucleotide sequence (SEQ ID NO:611) of a nativesequence PRO4993 cDNA, wherein SEQ ID NO:611 is a clone designatedherein as “DNA94832-2659”.

FIG. 230 shows the amino acid sequence (SEQ ID NO:612) derived from thecoding sequence of SEQ ID NO:611 shown in FIG. 229.

FIG. 231 shows a nucleotide sequence (SEQ ID NO:613) of a nativesequence PRO1559 cDNA, wherein SEQ ID NO:613 is a clone designatedherein as “DNA68886”.

FIG. 232 shows the amino acid sequence (SEQ ID NO:614) derived from thecoding sequence of SEQ ID NO:613 shown in FIG. 231.

FIG. 233 shows a nucleotide sequence (SEQ ID NO:615) of a nativesequence PRO725 cDNA, wherein SEQ ID NO:615 is a clone designated hereinas “DNA52758-1399”.

FIG. 234 shows the amino acid sequence (SEQ ID NO:616) derived from thecoding sequence of SEQ ID NO:615 shown in FIG. 233.

FIG. 235 shows a nucleotide sequence (SEQ ID NO:617) of a nativesequence PRO739 cDNA, wherein SEQ ED NO:617 is a clone designated hereinas “DNA52756”.

FIG. 236 shows the amino acid sequence (SEQ ID NO:618) derived from thecoding sequence of SEQ ID NO:617 shown in FIG. 235.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are comtemplated by the present invention.

The approximate location of the '“signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1–6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683–4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 81% amino acid sequence identity,more preferably at least about 82% amino acid sequence identity, morepreferably at least about 83% amino acid sequence identity, morepreferably at least about 84% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 86% amino acid sequence identity, morepreferably at least about 87% amino acid sequence identity, morepreferably at least about 88% amino acid sequence identity, morepreferably at least about 89% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 91% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, morepreferably at least about 93% amino acid sequence identity, morepreferably at least about 94% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 96% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, morepreferably at least about 98% amino acid sequence identity and mostpreferably at least about 99% amino acid sequence identity with afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, often at least about 20 amino acids inlength, more often at least about 30 amino acids in length, more oftenat least about 40 amino acids in length, more often at least about 50amino acids in length, more often at least about 60 amino acids inlength, more often at least about 70 amino acids in length, more oftenat least about 80 amino acids in length, more often at least about 90amino acids in length, more often at least about 100 amino acids inlength, more often at least about 150 amino acids in length, more oftenat least about 200 amino acids in length, more often at least about 300amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460–480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389–3402 (1997)). NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. “PRO variant polynucleotide” or “PROvariant nucleic acid sequence” means a nucleic acid molecule whichencodes an active PRO polypeptide as defined below and which has atleast about 80% nucleic acid sequence identity with a nucleotide acidsequence encoding a full-length native sequence PRO polypeptide sequenceas disclosed herein, a full-length native sequence PRO polypeptidesequence lacking the signal peptide as disclosed herein, anextracellular domain of a PRO polypeptide, with or without the signalpeptide, as disclosed herein or any other fragment of a full-length PROpolypeptide sequence as disclosed herein. Ordinarily, a PRO variantpolynucleotide will have at least about 80% nucleic acid sequenceidentity, more preferably at least about 81% nucleic acid sequenceidentity, more preferably at least about 82% nucleic acid sequenceidentity, more preferably at least about 83% nucleic acid sequenceidentity, more preferably at least about 84% nucleic acid sequenceidentity, more preferably at least about 85% nucleic acid sequenceidentity, more preferably at least about 86% nucleic acid sequenceidentity, more preferably at least about 87% nucleic acid sequenceidentity, more preferably at least about 88% nucleic acid sequenceidentity, more preferably at least about 89% nucleic acid sequenceidentity, more preferably at least about 90% nucleic acid sequenceidentity, more preferably at least about 91% nucleic acid sequenceidentity, more preferably at least about 92% nucleic acid sequenceidentity, more preferably at least about 93% nucleic acid sequenceidentity, more preferably at least about 94% nucleic acid sequenceidentity, more preferably at least about 95% nucleic acid sequenceidentity, more preferably at least about 96% nucleic acid sequenceidentity, more preferably at least about 97% nucleic acid sequenceidentity, more preferably at least about 98% nucleic acid sequenceidentity and yet more preferably at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthnative sequence PRO polypeptide sequence as disclosed herein, afull-length native sequence PRO polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460–480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389–3402 (1997)). NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions, see Table 6 below). For purposes herein, the% value of positives is determined by dividing (a) the number of aminoacid residues scoring a positive value between the PRO polypeptide aminoacid sequence of interest having a sequence derived from the native PROpolypeptide sequence and the comparison amino acid sequence of interest(i.e., the amino acid sequence against which the PRO polypeptidesequence is being compared) as determined in the BLOSUM62 matrix ofWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest.

Unless specifically stated otherwise, the % value of positives iscalculated as described in the immediately preceding paragraph. However,in the context of the amino acid sequence identity comparisons performedas described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 6 below) of the amino acidresidue of interest.

For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the %value of positives of a given amino acid sequence A to, with, or againsta given amino acid sequence B (which can alternatively be phrased as agiven amino acid sequence A that has or comprises a certain % positivesto, with, or against a given amino acid sequence B) is calculated asfollows:100 times the fraction X/Ywhere X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 or NCBI-BLAST2in that program's alignment of A and B, and where Y is the total numberof amino acid residues in B. It will be appreciated that where thelength of amino acid sequence A is not equal to the length of amino acidsequence B, the % positives of A to B will not equal the % positives ofB to A.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used inthe broadest sense and specificallycovers, for example, single anti-PRO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-PRO antibodycompositions with polyepitopic specificity, single chain anti-PROantibodies, and fragments of anti-PRO antibodies (see below). The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37–50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057–1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimun antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269–315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444–6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A 'small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

As used herein, “vascular endothelial cell growth factor-E,” or“VEGF-E,” refers to a mammalian growth factor as described herein,including the human amino acid sequence of FIG. 207, a sequence whichhas homology to VEGF and bone morphogenetic protein 1 and which includescomplete conservation of all VEGF cysteine residues, which have beenshown to be required for biological activity of VEGF. VEGF-E expressionincludes expression in human fetal bone, thymus, and thegastrointestinal tract. The biological activity of native VEGF-E isshared by any analogue or variant thereof that is capable of promotingselective growth and/or survival of umbilical vein endothelial cells,induces proliferation of pluripotent fibroblast cells, induces immediateearly gene c-fos in human endothelial cell lines and causes myocytehypertrophy in cardiac cells, or which possesses an immune epitope thatis immunologically cross-reactive with an antibody raised against atleast one epitope of the corresponding native VEGF-E. The human VEGF-Eherein is active on rat and mouse cells indicating conservation acrossspecies. Moreover, the VEGF-E herein is expressed at the growth plateregion and has been shown to embrace fetal myocytes.

As used herein, “vascular endothelial cell growth factor,” or “VEGF,”refers to a mammalian growth factor as defined in U.S. Pat. No.5,332,671. The biological activity of native VEGF is shared by anyanalogue or variant thereof that is capable of promoting selectivegrowth of vascular endothelial cells but not of bovine cornealendothelial cells, lens epithelial cells, adrenal cortex cells, BHK-21fibroblasts, or keratinocytes, or that possesses an immune epitope thatis immunologically cross-reactive with an antibody raised against atleast one epitope of the corresponding native VEGF.

The terms “VEGF-E polypeptide” and “VEGF-E” when used herein encompassnative sequence VEGF-E polypeptide and VEGF-E polypeptide variants(which are further defined herein). The VEGF-E polypeptides may beisolated from a variety of sources, such as from human tissue types orfrom another source, or prepared by recombinant or synthetic methods.

Inhibitors of VEGF-E include those which reduce or inhibit the activityor expression of VEGF-E and includes antisense molecules.

The abbreviation “KDR” refers to the kinase domain region of the VEGFmolecule. VEGF-E has no homology with VEGF in this domain.

The abbreviation “FLT-1” refers to the FMS-like tyrosine kinase bindingdomain which is known to bind to the corresponding FLT-1 receptor.VEGF-E has no homology with VEGF in this domain.

“Toll receptor2”, “TLR2” and “huTLR2” are used interchangeably, andrefer to a human Toll receptor designated as “HuTLR2” by Rock et al.,Proc. Natl. Acad. Sci. USA 95, 588–593 (1998).

The term “lipopolysaccharide” or “LPS” is used herein as a synonym of“endotoxin.” Lipopolysaccharides (LPS) are characteristic components ofthe outer membrane of Gram-negative bacteria, e.g., Escherichia coli.They consist of a polysaccharide part and a fat called lipid A. Thepolysaccharide, which varies from one bacterial species to another, ismade up of the O-specific chain (built from repeating units of three toeight sugars) and the two-part core. Lipid A virtually always includestwo glucosamine sugars modified by phosphate and a variable number offatty acids. For further information see, for example, Rietschel andBrade, Scientific American August 1992, 54–61.

The term 'septic shock” is used herein in the broadest sense, includingall definitions disclosed in Bone, Ann. Intern Med. 114, 332–333 (1991).Specifically, septic shock starts with a systemic response to infection,a syndrome called sepsis. When this syndrome results in hypotension andorgan dysfunction, it is called septic shock. Septic shock may beinitiated by gram-positive organisms and fungi, as well asendotoxin-containing Gram-negative organisms. Accordingly, the presentdefinition is not limited to “endotoxin shock.”

The phrases “gene amplification” and “gene duplication” are usedinterchangeably and refer to a process by which multiple copies of agene or gene fragment are formed in a particular cell or cell line. Theduplicated region (a stretch of amplified DNA) is often referred to as“amplicon”. Usually, the amount of the messenger RNA (mRNA) produced,i.e., the level of gene expression, also increases in the proportion ofthe number of copies made of the particular gene expressed.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer” and “cancerous” refer toor describe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include breastcancer, prostate cancer, colon cancer, squamous cell cancer, small-celllung cancer, non-small cell lung cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I131,I₁₂₅, Y90 and Re186), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g. paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere<<, Rhone-Poulenc Rorer,Antony, France), toxotere, methotrexate, cisplatin, melphalan,vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C,mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide,daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins,esperamicins (see U.S. Pat. No. 4,675,187), melphalan and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors such astamoxifen and onapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al. (W BSaunders: Philadelphia, 1995), especially p. 13.

“Doxorubicin” is an athracycline antibiotic.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; and the like. As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture and biologically active equivalents of the native sequencecytokines.

TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M   −8   /* value of a match with a stop */ int  _day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V W X YZ */ /* A */  {2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2, 1,1, 0, 0,−6, 0,−3, 0}, /* B */  {0, 3,−4, 3, 2,−5, 0, 1,−2, 0, 0,−3,−2,2,_M,−1, 1, 0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */ {−2,−4,15,−5,−5,−4,−3,−3,−2, 0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8,0, 0,−5}, /* D */  {0, 3,−5, 4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1,2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /* E */  {0, 2,−5, 3, 4,−5, 0, 1,−2, 0,0,−3,−2, 1,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 3}, /* F */ {−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2, 0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0,0, 7,−5}, /* G */  {1, 0,−3, 1, 0,−5, 5,−2,−3, 0,−2,−4,−3,0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */  {−1, 1,−3, 1, 1,−2,−2,6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3, 0, 0, 2}, /* I */ {−1,−2,−2,−2, 2, 1,−3,−2, 5, 0,−2, 2, 2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5,0,−1,−2}, /* J */  {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */  {−1, 0,−5, 0, 0,−5,−2, 0,−2, 0,5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3, 0,−4, 0}, /* L */ {−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6, 4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2,0,−1,−2}, /* M */  {−1,−2,−5,−3, 2, 0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1,0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */  {0, 2,−4, 2, 1,−4, 0, 2,−2, 0,1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4, 0,−2, 1}, /* O */ {_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */  {1,−1,−3,−1,−1,−5,−1,0,−2, 0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */  {0,1,−5, 2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4,3}, /* R */  {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6,0,−1, 0,−2, 2, 0,−4, 0}, /* S */  {1, 0, 0, 0, 0,−3, 1,−1,−1, 0,0,−3,−2, 1,_M, 1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */  {1, 0,−2, 0,0,−3, 0,−1, 0, 0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U*/  {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0}, /* V */  {0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2,2,−2,_M,−1,−2,−2,−1, 0, 0, 4,−6, 0,−2,−2}, /* W */  {−6,−5,−8,−7,−7,0,−7,−3,−5, 0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */ {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* Y */  {−3,−3, 0,−4,−4, 7,−5, 0,−1,0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2, 0, 0,10,−4}, /* Z */  {0, 1,−5,2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0, 3, 0, 0, 0, 0,−2,−6, 0,−4, 4} };/*  */ #include <stdio.h> #include <ctype.h> #define MAXJMP 16 /* maxjumps in a diag */ #define MAXGAP 24 /* don't continue to penalize gapslarger than this */ #define JMPS 1024 /* max jmps in an path */ #defineMX 4 /* save if there's at least MX-1 bases since last jmp */ #defineDMAT 3 /* value of matching bases */ #define DMIS 0 /* penalty formismatched bases */ #define DINS0 8 /* penalty for a gap */ #defineDINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for a gap */#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP];/* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no.of jmp in seq x */ }; /* limits seq to 2{circumflex over ( )}16 −1 */struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name for errmsgs */ char *seqx[2];   /* seqs: getseqs( ) */ int dmax; /* best diag:nw( ) */ int dmax0; /* final diag */ int dna; /* set if dna: main( ) */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2] /* holds pathfor seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char*getseq( ), *g_calloc( ); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  *  where file1 and file2 are two dna or twoprotein sequences.  *  The sequences can be in upper- or lower-case anmay contain ambiguity  *  Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  *  Max file length is 65535 (limited by unsigned short x in thejmp struct)  *  A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  *  Output is in the file “align.out”  *  * Theprogram may create a tmp file in /tmp to hold info about traceback.  *Original version developed under BSD 4.3 on a vax 8650  */ #include“nw.h” #include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1<<(‘D’-‘A’))|(1<<(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16,1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24,1<<25|(1<<(‘E’-‘A’))|(1<<(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,“usage: %s file1file2\n”, prog); fprintf(stderr,“where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr,“The sequences can be inupper- or lower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr,“Output is in thefile\”align.out\“\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw( ); /* fill in the matrix, getthe possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main( )  * dna: values in Fitch andSmith, PNAS, 80, 1382–1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw( ) nw { char *px, *py;    /* seqs and ptrs */ int *ndely, *dely;/* keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy < = len1; yy++) { col0[yy] =dely[yy] = col0[yy-1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy < = len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx < =len0; px,++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy < = len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 > =dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy] ++; } } else { if (col0[yy] − (ins0+ins1) >= dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if(col1[yy−1] − ins0 > =delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1yy−1] − (ins0+ins1) >= delx) { delx −col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis > = delx && mis >= dely[yy])col1[yy] = mis; else if (delx > = dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx > = MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij > = MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] > = MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].scoreDINS0)) { dx[id].ijmp++; if (++ij > = MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset + =sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = −ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy); if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps && xx< len0) col1[yy−1] −= ins0+ins1*(len0−xx); if(col1[yy−1] > smax) { smax= col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; } (void)free((cbar *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print( ) -- only routinevisible outside this module  *  * static:  * getmat( ) -- trace backbest path, count matches: print( )  * pr_align( ) -- print alignment ofdescribed in array p[ ]: print( )  * dumpblock( ) -- dump a block oflines with numbers, stars: pr_align( )  * nums( ) -- put out a numberline: dumpblock( )  * putline( ) -- put out a line (name, [num], seq,[num]): dumpblock( )  * stars( ) -- put a line of stars: dumpblock( )  *stripname( ) -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC 3 #define P_LINE 256 /* maximum output line */#define P_SPC 3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print( ) print { int   lx, ly, firstgap, lastgap;   /* overlap*/ if ((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “%s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) {   /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) {  /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align( ); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while(*p0 && *p1 ){ if (siz0) { p1++; n1++;siz0--; } else if (siz1) { p0++; n0++; siz1--; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “ ” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”:“residue”, (ngapx == 1)? “ ”:“s”); fprintf(fx,“%s”, outx);fprintf(fx, “,gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “ (%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy ==1)? “ ”:“s”); fprintf(fx, “%s”, outx); } if (dna) fprintf(fx, “/n<score:%d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “/n <score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized, left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “ ” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “” : “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars( ) */ /*  * print alignment of described in struct path pp[ ] */ static pr_align( ) pr_align { int nn; /* char count */ int more;register i; for (i = 0, lmax = 0; i < 2; i++) { nn =stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1;siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn = nm = 0,more = 1; more;) { ...pr_align for (i = more = 0; i < 2; i++) { /*  * dowe have more of this sequence?  */ if (!*ps[i]) continue; more++; if(pp[i].spc) {   /* leading space */ *po[i]++ =‘ ’; pp[i].spc−−; } elseif (siz[i]) {  /* in a gap */ *po[i]++ =‘−’; siz[i]−−; } else { /* we'reputting a seq element  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align( )  */ static dumpblock( ) dumpblock { register  i; for(i = 0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx);for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) !=‘ ’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars( );putline(i); if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1)nums(i); } } } /*  * put out a number line: dumpblock( )  */ staticnums(ix) nums int ix; /* index in out[ ] holding seq line */ { charnline[P_LINE]; register i, j; register char *pn, *px, *py; for (pn =nline, i = 0; i < lmax+P_SPC; i++, pn++) *pn = ‘ ’; for (i = nc[ix], py= out[ix]; *py; py++, pn++) { if (*py == ‘ ’ || *py == ‘−’) *pn = ‘ ’;else { if (i%10 == 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i;for (px = pn; j; j / = 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘−’;} else *pn = ‘ ’; i++; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline; *pn;pn++) (void) putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put out aline (name, [num], seq, [num]): dumpblock( )  */ static putline(ix)putline int ix; { int i; ...putline register char *px; for (px =namex[ix], i = 0; *px && *px != ‘:’; px++, i++) (void) putc(*px, fx);for(; i < lmax+P_SPC; i++) (void) putc(‘ ’, fx); /* these count from 1: * ni[ ] is current element (from 1)  * nc[ ] is number at start ofcurrent line  */ for (px = out[ix]; *px; px++) (void) putc(*px&0x7F,fx); (void) putc(‘\n’, fx); } /*  * put a line of stars (seqs always inout[0], out[1]): dumpblock( )  */ static stars( ) stars { int i;register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0] == ‘ ’ &&*(po[0]) == ‘ ’) ||   !*out[1] || (*out[1] == ‘ ’ && *(po[1]) == ‘ ’))return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for (p0 =out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align( )  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup( )−−cleanup any tmp file  * getseq( ) −−read in seq, set dna, len, maxlen * g_calloc( ) −−calloc( ) with error checkin  * readjmps( ) −−get thegood jmps, from tmp file if necessary  * writejmps( ) −−write a filledarray of jmps to a tmp file: nw( )  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) {fprintf(stderr, “%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, “%s: malloc( )failed to get %d bytes for %s\n”, prog, tlen+6, file); exit(1); }pseq[0] = pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4;*len = tlen; rewind(fp); while (fgets(line, 1024, fp)) { if(*line == ‘;’|| *line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”,*(py−1))) natgc++; } } *py++ = ‘\0’; *py= ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc( ) failed %s (n=%d, sz=%d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[ ] or tmp file, set pp[ ], reset dmax: main( )  */ readjmps( )readjmps { int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) {(void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {fprintf(stderr, “%s: can't open( ) %s\n”, prog, jname); cleanup(1); } }for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for(j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmpsif (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if(j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax + =siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1]= −siz; xx + =siz; /* id = xx − yy + len1 − 1 */ pp[1].x[i1] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; i1++; } else if (siz > 0) { /* gapin first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx + =siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz: MAXGAP; i0 ++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i, i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i, } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i, i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i0] = i, } if (fd > = 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw( ) */writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp( ) %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 =33.3%II. Compositions and Methods of the Invention

A. Full-Length PRO Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding various PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

1. Full-Length PRO213 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO213. In particular, Applicants have identified and isolated cDNAencoding a PRO213 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a portion of the PRO213 polypeptide hassignificant homology with the human growth arrest-specific 6 (gas6)protein. Accordingly, it is presently believed that PRO213 polypeptidedisclosed in the present application may have the same or simularactivity as does the gas6 protein.

2. Full-Length PRO274 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO274. In particular, Applicants have identified and isolated cDNAencoding a PRO274 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO274polypeptide have significant homology with the 7 transmembrane segmentreceptor proteins and Fn54 protein. Accordingly, it is presentlybelieved that PRO274 polypeptide disclosed in the present application isa newly identified member of the 7 transmembrane segment receptorprotein and/or Fn54 protein family.

3. Full-Length PRO300 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO300. In particular, Applicants have identified and isolated cDNAencoding a PRO300 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO300polypeptide have significant homology with the human Diff 33 protein.Accordingly, it is presently believed that PRO300 polypeptide disclosedin the present application is a newly identified member of the Diff 33family.

4. Full-Length PRO284 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO284. In particular, Applicants have identified and isolated cDNAencoding a PRO284 polypeptide, as disclosed in further detail in theExamples below. To Applicants present knowledge, the UNQ247(DNA23318-1211) nucleotide sequence encodes a novel factor; using BLASTand FastA sequence alignment computer programs, no sequence identitiesto any known proteins were revealed.

5. Full-Length PRO296 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO296. In particular, Applicants have identified and isolated cDNAencoding a PRO296 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO296 polypeptide has significantsimilarity to the sarcoma-amplified SAS protein. Accordingly, it ispresently believed that PRO296 polypeptide disclosed in the presentapplication is a newly identified SAS protein homolog.

6. Full-Length PRO329 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO329. In particular, Applicants have identified and isolated cDNAencoding a PRO329 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO329 polypeptide has significantsimilarity to a high affinity inmmunoglobulin F_(c) receptor.Accordingly, it is presently believed that PRO329 polypeptide disclosedin the present application is a newly identified F_(c) receptor homolog.

7. Full length PRO362 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO362. In particular, Applicants have identified and isolated cDNAencoding a PRO362 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO362 polypeptide has significantsimilarity to the A33 antigen protein as well as the HCAR protein andthe NrCAM related cell adhesion molecule. Accordingly, it is presentlybelieved that PRO362 polypeptide disclosed in the present application isa newly A33 antigen and HCAR protein homolog.

8. Full-Length PRO363 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO363. In particular, Applicants have identified and isolated cDNAencoding a PRO363 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO363 polypeptide has significantsimilarity to the cell surface protein HCAR. Accordingly, it ispresently believed that PRO363 polypeptide disclosed in the presentapplication is a newly HCAR homolog.

9. Full-Length PRO868 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO868. In particular, Applicants have identified and isolated cDNAencoding a PRO868 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO868 polypeptide has significantsimilarity to the tumor necrosis factor receptor. Accordingly, it ispresently believed that PRO868 polypeptide disclosed in the presentapplication is a newly identified member of the tumor necrosis factorreceptor family of proteins.

10. Full-Length PRO382 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO382. In particular, Applicants have identified and isolated cDNAencoding a PRO382 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the native PRO382 polypeptide sharessignificant homology with various serine protease proteins. Applicantshave also found that the DNA encoding the PRO382. polypeptide sharessignificant homology with nucleic acid encoding various serine proteaseproteins. Accordingly, it is presently believed that PRO382 polypeptidedisclosed in the present application is a newly identified serineprotease homolog.

11. Full-Length PRO545 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO545. In particular, Applicants have identified and isolated cDNAencoding a PRO545 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO545polypeptide have significant homology with the sequences identifieddesignated as; human metalloproteinase (“P_W01825”), mouse meltrin alpha(“S60257”), metalloprotease-disintegrin meltrin-alpha (“GEN13695 ”),ADAM 13—Xenopus laevis (“XLU66003_(—)1”), mouse meltrin beta (“S60258”),rabbit metalloprotease-disintegrin meltrin-beta, (“GEN13696”), humanmeltrin S (“AF023477_(—)1”), human meltrin precursor (“AF023476_(—)1”),human ADAM 21 (“AF029900_(—)1”), and human ADAM 20 (“AF0298991_(—)1”),thereby indicating that PRO545 may be a novel meltrin protein.Accordingly, it is presently believed that the PRO545 polypeptidedisclosed in the present application is a newly identified member of themeltrin family and possesses the cellular adhesiveness typical of themeltrin proteins which comprise both metalloprotease and disintegrindomains.

12. Full-Length PRO617 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO617. In particular, Applicants have identified and isolated cDNAencoding a PRO617 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO617 polypeptide sharessignificant homology with the CD24 protein. Applicants have also foundthat the DNA encoding the PRO617 polypeptide has significant homologywith DNA encoding the CD24 protein. Accordingly, it is presentlybelieved that PRO617 polypeptide disclosed in the present application isa newly identified CD24 homolog.

13. Full-Length PRO700 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO700. In particular, Applicants have identified and isolated cDNAencoding a PRO700 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO700 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO700 polypeptidepossess significant sequence similarity to various protein disulfideisomerases. More specifically, an analysis of the Dayhoff database(version 35.45 SwissProt 35) evidenced significant sequence similaritybetween the PRO700 amino acid sequence and the following Dayhoffsequences; polypeptide with protein disulfide isomerase activity,designated as (“P_P80664”), human PDI, designated as (“P_R51696”), humanPDI, designated as (P_R5297”), probable protein disulfide isomeraseer-60 precursor, designated as (“ER60_SCHMA”), protein disulfideisomerase precursor—Drosophila melanogaster, designated as(“PDI_DROME”), protein disulfide-isomerase precursor—Nicotiana tabaccum,designated as (“NTPDIGENE_(—)1”), protein disulfide isomerase—Onchocercavolvulus, designated as (“OVU12440_(—)1”), human probable proteindisulfide isomerase p5 precursor, designated as (“ERP5_HUMAN”), humanprotein disulfide isomerase-related protein 5, (“HSU79278_(—)1”), andprotein disulfide isomerase precursor/prolyl 4 hydroxy, (“PDI_HUMAN”),thereby indicating that PRO700 may be a novel protein disulfideisomerase. Accordingly, it is presently believed that PRO700 polypeptidedisclosed in the present application is a newly identified member of theprotein disulfide isomerase family and possesses the ability to catalyzethe formation of disulfide bonds typical of the protein disulfideisomerase family.

14. Full-Length PRO702 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO702. In particular, Applicants have identified and isolated cDNAencoding a PRO702 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO702 polypeptide has significantsimilarity to the conglutinin protein. Accordingly, it is presentlybelieved that PRO702 polypeptide disclosed in the present application isa newly identified conglutinin homolog.

15. Full-Length PRO703 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO703. In particular, Applicants have identified and isolated cDNAencoding a PRO703 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO703 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO703 polypeptidepossess significant sequence similarity to the VLCAS protein, therebyindicating that PRO703 may be a novel VLCAS protein. More specifically,an analysis of the Dayhoff database (version 35.45 SwissProt 35)evidenced significant sequence similarity between the PRO703 amino acidsequence and the following Dayhoff sequences, human mRNA forvery-long-chain acyl-CoA, (“D88308”), rat MnRNA for very-long-chainacyl-CoA synthetase, (“D85100”), Mus musculs fatty acid transportprotein, (“MMU15976”), human very-long-chain acyl-CoA synthetase,(“D88308_(—)1”), Mus musculus very-long-chain acyl-CoA synthetase,(“AP033031_(—)1”), very-long-chain acyl-CoA synthetase—Ratts,(“D85100_(—)1”), rat long-chain fatty acid transport protein,(“FATP_RAT”), mouse long-chain fatty acid transport protein,(“FATP_MOUSE”), probable long-chain fatty acid transport protein,(“FAT1_YEAST”), and fatty acid transporter protein, (“CHY15839_(—)2”),thereby indicating that PRO703 may be a novel VLCAS. Accordingly, it ispresently believed that PRO703 polypeptide disclosed in the presentapplication is a newly identified member of the VLCAS family andpossesses the ability to facilitate the cellular transport of long andvery long chain fatty acids typical of the VLCAS family.

16. Full-Length PRO705 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO705. In particular, Applicants have identified and isolated cDNAencoding a PRO705 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO705 polypeptide has significantsimilarity to the K-glypican protein. Accordingly, it is presentlybelieved that PRO705 polypeptide disclosed in the present application isa newly identified member of the glypican family of proteoglycanproteins.

17. Full-Length PRO708 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO708. In particular, Applicants have identified and isolated cDNAencoding a PRO708 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO708 polypeptide has significanthomology with the aryl sulfatase proteins. Applicants have also foundthat the DNA encoding the PRO708 polypeptide has significant homologywith DNA encoding the aryl sulfatase proteins. Accordingly, it ispresently believed that PRO708 polypeptide disclosed in the presentapplication is a newly identified aryl sulfatase homolog.

18. Full-Length PRO320 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO320. In particular, Applicants have identified and isolated cDNAencoding a PRO320 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO320 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO320 polypeptide havesignificant homology to the fibulin protein. More specifically, ananalysis of the Dayhoff database (version 35.45 SwissProt 35) evidencedsignificant homology between the PRO320 amino acid sequence and thefollowing Dayhoff sequences, human fibulin-2 precursor, designated“FBL2_HUMAN”, human fibulin-1 isoform a precursor, designated“FBLA_HUMAN”, ZK783.1—Caenorhabditis elegans, designated“CELZK783_(—)1”, human-notcb2, designated “HSU77493_(—)1”, Nel proteinprecursor—rattus norvegicus, designated “NEL_RAT”, Mus musculus cellsurface protein, designated “D32210_(—)1”, mouse (fragment) Notch Bprotein, designated “A49175”, C50H2.3a—Caenorhabditis elegans,designated “CEC50H2_(—)3”, MEC-9L—Caenorhabditis elegans, designated“CEU33933_(—)1”, and Mus musculus notch 4, designated “10MMMHC29N7_(—)2”, thereby indicating that PRO320 may be a novel fibulinor fibulin-like protein. Accordingly, it is presently believed thatPRO320 polypeptide disclosed in the present application is a newlyidentified member of the fibulin family and possesses biologicalactivity typical of the fibulin family.

19. Full-Length PRO324 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO324. In particular, Applicants have identified and isolated cDNAencoding a PRO324 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO324 polypeptide has significantsimilarity to oxidoreductases. Accordingly, it is presently believedthat PRO324 polypeptide disclosed in the present application is a newlyidentified oxidoreductase homolog.

20. Full-Length PRO351 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO351. In particular, Applicants have identified and isolated cDNAencoding a PRO351 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO351 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO351 polypeptidepossess significant sequence similarity to the prostasin protein,thereby indicating that PRO351 may be a novel prostasin protein. Morespecifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant sequence similarity between thePRO351 amino acid sequence and the following Dayhoff sequences,“AC003965_(—)1”, “CELC07G1_(—)7”, “GEN12917”, “HEPS_HUMAN”, “GEN14584”,“MCT6_MOUSE”, “HSU75329_(—)1”, “PLMN_ERIEU”, “TRYB_HUMAN”, and“P_W22987”. Accordingly, it is presently believed that PRO351polypeptide disclosed in the present application is a newly identifiedmember of the prostasin family and possesses properties and activitiestypical of the prostasin family.

21. Full-Length PRO352 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO352. In particular, Applicants have identified and isolated cDNAencoding a PRO352 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO352 polypeptide has significantsimilarity to the butyrophilin protein. Accordingly, it is presentlybelieved that PRO352 polypeptide disclosed in the present application isa newly identified butyrophilin homolog.

22. Full-Length PRO381 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO381. In particular, Applicants have identified and isolated cDNAencoding a PRO381 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO381 polypeptide has significantsimilarity to immunophilin proteins. Accordingly, it is presentlybelieved that PRO381 polypeptide disclosed in the present application isa newly identified FKBP immunophilin homolog.

23. Full-Length PRO386 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO386. In particular, Applicants have identified and isolated cDNAencoding a PRO386 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO386polypeptide has significantsimilarity to the beta-2 subunit of a sodium channel protein.Accordingly, it is presently believed that PRO386 polypeptide disclosedin the present application is homolog of a beta-2 subunit of a sodiumchannel expressed in mammalian cells.

24. Full-Length PRO540 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO540. In particular, Applicants have identified and isolated cDNAencoding a PRO540 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO540 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO540 polypeptidepossess significant sequence similarity to the LCAT protein, therebyindicating that PRO540 may be a novel LCAT protein. More specifically,an analysis of the Dayhoff database (version 35.45 SwissProt 35)evidenced significant sequence similarity between the PRO540 amino acidsequence and the following Dayhoff sequences, phosphatidylcholine-sterolacyltransferase, designated “LCAT_HUMAN”, hypothetical 75.4 kd protein,designated “YN84_YEAST”, Bacillus licheniforms esterase, designated“BLU35855_(—)1”, macrotetrolide resistance protein—Streptomyces,designated “JH0655”, T-cell receptor delta chain precursor, designated“C30583”, Rhesus kringle 2, designated “P_W07551”, RAGE-1 ORF5,designated “HSU46191_(—)3”, human Ig kappa chain VKIII-JK3, designated“HSU07466_(—)1”, and Alstroemeria inodora reverse transcriptase,designated “AL1223606_(—)1”. Accordingly, it is presently believed thatPRO540 polypeptide disclosed in the present application is a newlyidentified member of the LCAT protein family and possesses lipidtransport capability typical of the LCAT family.

25. Full-Length PRO615 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO615. In particular, Applicants have identified and isolated cDNAencoding a PRO615 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO615 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO615 polypeptidepossess significant sequence similarity to the human synaptogyrinprotein, thereby indicating that PRO615 may be a novel synaptogyrinprotein. More specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35) evidenced significant sequence similarity betweenthe PRO615 amino acid sequence and the following Dayhoff sequences,“AF039085_(—)1”, “RNU39549_(—)1”, “CELT08A9_(—)8”, “FSU62028_(—)1”,“S73645”, “Y348_MYCPN”, “AC000103_(—)5”, “ ”, “RT12_LEITA”, and“EBVLMP218_(—)1”. Accordingly, it is presently believed that PRO615polypeptide disclosed in the present application is a newly identifiedmember of the synaptogyrin family and possesses activity and propertiestypical of the synaptogyrin family.

26. Full-Length PRO618 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO618. In particular, Applicants have identified and isolated cDNAencoding a PRO618 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO618 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO618 polypeptidepossess significant sequence similarity to the enteropeptidase protein,thereby indicating that PRO618 may be a novel enteropeptidase. Morespecifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant sequence similarity between thePRO618 amino acid sequence and the following Dayhoff sequences,“P_W22987”, “KAL_HUMAN”, “AC00395_(—)1”, “GEN12917”, “ENTK_HUMAN”,“FA11_HUMAN”, “HSU75329_(—)1”, “P_W22986”, and “PLMN_HORSE”.Accordingly, it is presently believed that PRO618 polypeptide disclosedin the present application is a newly identified member of theenteropeptidase family and possesses catalytic activity typical of theenteropeptidase family.

27. Full-Length PRO719 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO719. In particular, Applicants have identified and isolated cDNAencoding a PRO719 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO719 polypeptide has significantsimilarity to the lipoprotein lipase H protein. Accordingly, it ispresently believed that PRO719 polypeptide disclosed in the presentapplication is a newly identified lipoprotein lipase H homolog.

28. Full-Length PRO724 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO724. In particular, Applicants have identified and isolated cDNAencoding a PRO724 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO724 polypeptide has significantsimilarity to the human low density lipoprotein (LDL) receptor protein.Accordingly, it is presently believed that PRO724 polypeptide disclosedin the present application is a newly identified LDL receptor homolog.

29. Full-Length PRO772 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO772. In particular, Applicants have identified and isolated cDNAencoding a PRO772 polypeptide, as disclosed in fer detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO772 polypeptide has significantsimilarity to the human A4 protein. Accordingly, it is presentlybelieved that PRO772 polypeptide disclosed in the present application isa newly identified A4 protein homolog.

30. Full-Length PRO852 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO852. In particular, Applicants have identified and isolated cDNAencoding a PRO852 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO852 polypeptide has significantsimilarity to various protease proteins. Accordingly, it is presentlybelieved that PRO852 polypeptide disclosed in the present application isa newly identified protease enzyme homolog.

31. Full-Length PRO853 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO853. In particular, Applicants have identified and isolated cDNAencoding a PRO853 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO853 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO853 polypeptidepossess significant sequence similarity to the reductase protein,thereby indicating that PRO853 may be a novel reductase. Morespecifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant sequence similarity between thePRO853 amino acid sequence and the following Dayhoff sequences,“P_W03198”, “CEC15H11_(—)6”, “MTV030_(—)12”, “P_W15759”, “S42651”,“ATAC00234314”, “MTV022_(—)13”, “SCU437041”, “CELE04F6_(—)7”, and“ALFA_(—)1”. Accordingly, it is presently believed that PRO853polypeptide disclosed in the present application is a newly identifiedmember of the reductase family and possesses the antioxidant enzymaticactivity typical of the reductase family.

32. Full-Length PRO860 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO860. In particular, Applicants have identified and isolated cDNAencoding a PRO860 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO860 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO860 polypeptidepossess significant sequence similarity to the neurofascin protein,thereby indicating that PRO860 may be a novel neurofascin. Morespecifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant sequence similarity between thePRO860 amino acid sequence and the following Dayhoff sequences,“AF040990_(—)1”, “AF041053_(—)1”, “CELZK377_(—)2”, “RNU81035_(—)1”,“D86983_(—)1”, “S26180”, “MMBIG2A_(—)1”, “S46216”, and “RNU68726_(—)1”.Accordingly, it is presently believed that PRO860 polypeptide disclosedin the present application is a newly identified member of theneurofascin family and possesses the cellular adhesion propertiestypical of the neurofascin family.

33. Full-Length PRO846 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO846. In particular, Applicants have identified and isolated cDNAencoding a PRO846 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO846 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO846 polypeptidepossess significant sequence similarity to the CMRF35 protein, therebyindicating that PRO846 may be a novel CMRF35 protein. More specifically,an analysis of the Dayhoff database (version 35.45 SwissProt 35)evidenced significant sequence similarity between the PRO846 amino acidsequence and the following Dayhoff sequences, “CM35_HUMAN”,“AF035963_(—)1”, “PIGR_RABIT”, “AF043724_(—)1”, “RNU89744_(—)1”,“A52091_(—)1”, “S48841”, “ELK06A9_(—)3”, and “AF049588_(—)1”.Accordingly, it is presently believed that PRO846 polypeptide disclosedin the present application is a newly identified member of the CMRF35protein family and possesses properties typical of the CMRF35 proteinfamily.

34. Full-Length PRO862 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO862. In particular, Applicants have identified and isolated cDNAencoding a PRO862 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO862 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO862 polypeptidepossess significant sequence similarity to the lysozyme protein, therebyindicating that PRO862 may be a novel lysozyme protein. Morespecifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant sequence similarity between thePRO862 amino acid sequence and the following Dayhoff sequences,“P_P90343”, and “LYC_HUMAN”. Accordingly, it is presently believed thatPRO862 polypeptide disclosed in the present application is a newlyidentified member of the lysozyme family and possesses catalyticactivity typical of the lysozyme family.

35. Full-Length PRO864 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO864. In particular, Applicants have identified and isolated cDNAencoding a PRO864 polypeptide, as disclosed in further detail in theExamples below. Analysis of the amino acid sequence of the full-lengthPRO864 polypeptide using BLAST and FastA sequence alignment computerprograms, suggests that various portions of the PRO864 polypeptidepossess significant sequence similarity to the Wnt-4 protein, therebyindicating that PRO864 may be a novel Wnt-4 protein. More specifically,an analysis of the Dayhoff database (version 35.45 SwissProt 35)evidenced significant sequence similarity between the PRO864 amino acidsequence and the following Dayhoff sequences, “WNT4_MOUSE”,“WNT3_MOUSE”, “WN5A_HUMAN”, “WN7B_MOUSE”, “WN3A_MOUSE”, “XLU66288_(—)1”,“WN13_HUMAN”, “WN5B_ORYLA”, “WNT2_MOUSE”, and “WN7A_MOUSE”. Accordingly,it is presently believed that PRO864 polypeptide disclosed in thepresent application is a newly identified member of the Wnt-4 proteinfamily and possesses properties typical of the Wnt-4 protein family.

36. Full-Length PRO792 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO792. hI particular, Applicants have identified and isolated cDNAa, encoding a PRO792 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO792 polypeptide has significantsimilarity to the CD23 protein. Accordingly, it is presently believedthat PRO792 polypeptide disclosed in the present application is a newlyidentified CD23 homolog.

37. Full-Length PRO866 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO866. In particular, Applicants have identified and isolated cDNAencoding a PRO866 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO866 polypeptide has significantsimilarity to various mindin and spondin proteins. Accordingly, it ispresently believed that PRO866 polypeptide disclosed in the presentapplication is a newly identified mindin/spondin homolog.

38. Full-Length PRO871 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO871. Ia particular, Applicants have identified and isolated cDNAencoding a PRO871 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO871 polypeptide has significantsimilarity to the CyP-60 protein. Accordingly, it is presently believedthat PRO871 polypeptide disclosed in the present application is a newlyidentified member of the cyclophilin protein family and possessesactivity typical of the cyclophilin protein family.

39. Full-Length PRO873 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO873. In particular, Applicants have identified and isolated cDNAencoding a PRO873 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO873 polypeptide has significantsimilarity to a human liver carboxylesterase. Accordingly, it ispresently believed that PRO873 polypeptide disclosed in the presentapplication is a newly identified member of the carboxylesterase familyand possesses enzymatic activity typical of the carboxylesterase family.

40. Full-Length PRO940 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO940. In particular, Applicants have identified and isolated cDNAencoding a PRO940 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO940 polypeptide has significantsimilarity to CD33 and the OB binding protein-2. Accordingly, it ispresently believed that PRO940 polypeptide disclosed in the presentapplication is a newly CD33 and/or OB binding protein-2 homolog.

41. Full-Length PRO941 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO941. In particular, Applicants have identified and isolated cDNAencoding a PRO941 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO941 polypeptide has significantsimilarity to one or more cadherin proteins. Accordingly, it ispresently believed that PRO941 polypeptide disclosed in the presentapplication is a newly identified cadherin homolog.

42. Full-Length PRO944 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO944. In particular, Applicants have identified and isolated cDNAencoding a PRO944 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO944 polypeptide has significantsimilarity to the CPE-R cell surface protein. Accordingly, it ispresently believed that PRO944 polypeptide disclosed in the presentapplication is a newly identified CPE-R homolog.

43. Full-Length PRO983 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO983. In particular, Applicants have identified and isolated cDNAencoding a PRO983 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO983 polypeptide has significantsimilarity to the vesicle-associated protein, VAP-33. Accordingly, it ispresently believed that PRO983 polypeptide disclosed in the presentapplication is a newly identified member of the vesicle-associatedmembrane protein family and possesses activity typical ofvesicle-associated membrane proteins.

44. Full-Length PRO1057 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1057. In particular, Applicants have identified and isolated cDNAencoding a PRO1057 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO1057 polypeptide has significantsimilarity to various protease proteins. Accordingly, it is presentlybelieved that PRO1057 polypeptide disclosed in the present applicationis a newly identified protease homolog.

45. Full-Length PRO1071 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1071. In particular, Applicants have identified and isolated cDNAencoding a PRO1071 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO1071 polypeptide has significantsimilarity to the thrombospondin protein. Accordingly, it is presentlybelieved that PRO1071 polypeptide disclosed in the present applicationis a newly identified thrombospondin homolog.

46. Full-Length PRO1072 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1072. In particular, Applicants have identified and isolated cDNAencoding a PRO1072 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO1072 polypeptide has significantsimilarity to various reductase proteins. Accordingly, it is presentlybelieved that PRO1072 polypeptide disclosed in the present applicationis a newly identified member of the reductase protein family.

47. Full-Length PRO1075 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1075. In particular, Applicants have identified and isolated cDNAencoding a PRO1075 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO1075 polypeptide has significantsimilarity to protein disulfide isomerase. Accordingly, it is presentlybelieved that PRO1075 polypeptide disclosed in the present applicationis a newly identified member of the protein disulfide isomerase familyand possesses activity typical of that family.

48. Full-Length PRO181 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO181. In particular, Applicants have identified and isolated cDNAencoding a PRO181 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO181 polypeptide has significantsimilarity to the cornichon protein. Accordingly, it is presentlybelieved that PRO181 polypeptide disclosed in the present application isa newly identified cornichon homolog.

49. Full-Length PRO195 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO195. In particular, Applicants have identified and isolated cDNAencoding a PRO195 polypeptide, as disclosed in further detail in theExamples below. The PRO195-encoding clone was isolated from a humanfetal placenta library using a trapping technique which selects fornucleotide sequences encoding secreted proteins. To Applicants presentknowledge, the UNQ169 (DNA26847-1395) nucleotide sequence encodes anovel factor; using BLAST and FastA sequence alignment computerprograms, no sequence identities to any known proteins were revealed.

50. Full-Length PRO865 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO865. In particular, Applicants have identified and isolated cDNAencoding a PRO865 polypeptide, as disclosed in further detail in theExamples below. The PRO865-encoding clone was isolated from a humanfetal kidney library using a trapping technique which selects fornucleotide sequences encoding secreted proteins. Thus, thePRO865-encoding clone may encode a secreted factor. To Applicantspresent knowledge, the UNQ434 (DNA53974-1401) nucleotide sequenceencodes a novel factor; using BLAST and FastA sequence alignmentcomputer programs, no sequence identities to any known proteins wererevealed.

51. Full-Length PRO827 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO827. In particular, Applicants have identified and isolated cDNAencoding a PRO827 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO827 polypeptide has significantsimilarity to VLA-2 and various other integrin proteins. Accordingly, itis presently believed that PRO827 polypeptide disclosed in the presentapplication is a novel integrin protein or splice variant thereof.

52. Full-Length PRO1114 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1114. In particular, Applicants have identified and isolated cDNAencoding a PRO1114 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO1114 polypeptide has significantsimilarity to the cytokine receptor family of proteins. Accordingly, itis presently believed that PRO1114 polypeptide disclosed in the presentapplication is a newly identified member of the cytokine receptor familyof proteins and possesses activity typical of that family.

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1114 interferon receptor (UNQ557). In particular, cDNA encoding aPRO1114 interferon receptor polypeptide has been identified andisolated, as disclosed in further detail in the Examples below. It isnoted that proteins produced in separate expression rounds may be givendifferent PRO numbers but the UNQ number is unique for any given DNA andthe encoded protein, and will not be changed. However, for sake ofsimplicity, in the present specification the protein encoded byDNA57033-1403 as well as all further native homologues and variantsincluded in the foregoing definition of PRO1114 interferon receptor,will be referred to as “PRO1114 interferon receptor”, regardless oftheir origin or mode of preparation.

Using the WU-BLAST2 sequence alignment computer program, it has beenfound that a full-length native sequence PRO1114 interferon receptorpolypeptide (shown in FIG. 142 and SEQ ID NO:352) has sequence identitywith the other known interferon receptors. Accordingly, it is presentlybelieved that PRO1114 interferon receptor possesses activity typical ofother interferon receptors.

53. Full-Length PRO237 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO237. In particular, Applicants have identified and isolated cDNAencoding a PRO237 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO237 polypeptide has significantsimilarity to carbonic anhydrase. Accordingly, it is presently believedthat PRO237 polypeptide disclosed in the present application is a newlyidentified carbonic anhydrase homolog.

54. Full-Length PRO541 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO541. In particular, Applicants have identified and isolated cDNAencoding a PRO541 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO541 polypeptide has significantsimilarity to a trypsin inhibitor protein. Accordingly, it is presentlybelieved that PRO541 polypeptide disclosed in the present application isa newly identified member of the trypsin inhibitor protein family.

55. Full-Length PRO273 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO273. In particular, Applicants have identified and isolated cDNAencoding a PRO273 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO273polypeptide have significant sequence identity with various chemokines.Accordingly, it is presently believed that PRO273 polypeptide disclosedin the present application is a newly identified member of the chemokinefamily and possesses activity typical of the chemokine family.

56. Full-Length PRO701 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO701. In particular, Applicants have identified and isolated cDNAencoding a PRO701 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO701polypeptide have significant homology with the neuroligins 1, 2 and 3and esterases including carboxyesterases and acytlcholinesterases.Accordingly, it is presently believed that PRO701 polypeptide disclosedin the present application is a newly identified member of theneuroligin family and is involved in mediating recognition processesbetween neurons and/or functions as a cell adhesin molecule as istypical of neuroligins.

57. Full-Length PRO704 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO704. In particular, Applicants have identified and isolated cDNAencoding a PRO704 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO704polypeptide have significant homology with the VIP36 and GP36b.Accordingly, it is presently believed that PRO704 polypeptide disclosedin the present application is a newly identified member of the vesicularintegral membrane protein family and possesses the ability to bind tosugars and cycle between the plasma membrane and the Golgi typical ofthis family.

58. Full-Length PRO706 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO706. In particular, Applicants have identified and isolated cDNAencoding a PRO706 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO706polypeptide have sequence identity with the human prostatic acidphosphatase precursor and the human lysosomal acid phosphataseprecursor. Accordingly, it is presently believed that PRO706 polypeptidedisclosed in the present application is a newly identified member of thehuman prostatic acid phosphatase precursor family and possessesphosphatase typical of the acid phosphatase family.

59. Full-Length PRO707 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO707. In particular, Applicants have identified and isolated cDNAencoding a PRO707 polypeptide, as disclosed in futher detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO707polypeptide have significant homology with cadherins, particularlycadherin FIB3 found in fibroblasts. Accordingly, it is presentlybelieved that PRO707 polypeptide disclosed in the present application isa newly identified member of the cadherin family and possesses cellinteraction signaling typical of the cadherin family.

60. Full-Length PRO322 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO322. In particular, Applicants have identified and isolated cDNAencoding a PRO322 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO322polypeptide have significant homology with human neuropsin, serineprotease, neurosin and trypsinogen. Accordingly, it is presentlybelieved that PRO322 polypeptide disclosed in the present application isa newly identified member of the serine protease family and possessesprotease activity typical of this family. It is also believed thatPRO322 is involved in hippocampal plasticity and is associated withextracellular matrix modifications and cell migrations.

61. Full-Length PRO526 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO526. In particular, Applicants have identified and isolated cDNAencoding a PRO526 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO526polypeptide have significant homology with the acid labile subunit ofthe insulin-like growth factor complex (ALS), as well carboxypeptidase,SLIT, and platelet glycoprotein V. Accordingly, it is presently believedthat PRO526 polypeptide disclosed in the present application is a newlyidentified member of the leucine-repeat rich superfamily, and possessesprotein-protein interaction capabilities typical of this family.

62. Full-Length PRO531 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO531. In particular, Applicants have identified and isolated cDNAencoding a PRO531 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO531polypeptide have significant sequence identity and similarity withmembers of the cadherin superfamily, particularly, protocadherinAccordingly, it is presently believed that PRO531 polypeptide disclosedin the present application is a newly identified member of the cadherinsuperfamily, and is a protocadherin. PRO531 is a transmembrane proteinwhich has extracellular cadherin motifs. PRO531 is believed to beinvolved in cell-cell activity, in particular, cell signaling.

63. Full-Length PRO534 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO534. In particular, Applicants have identified and isolated cDNAencoding a PRO534 polypeptide, as disclosed in futher detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO534polypeptide have significant identity or similarity with the putativedisulfide isomerase erp38 precursor and thioredoxin c-3. Accordingly, itis presently believed that PRO534 polypeptide disclosed in the presentapplication is a newly identified member of the disulfide isomerasefamily and possesses the ability to recognize and unscramble eitherintermediate or incorrect folding patterns typical of this family.

64. Full-Length PRO697 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO697. In particular, Applicants have identified and isolated cDNAencoding a PRO697 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO697polypeptide have significant identity or similarity with sFRP-2, sFRP-1and SARP-1, -2 and -3. Accordingly, it is presently believed that PRO697polypeptide disclosed in the present application is a newly identifiedmember of the sFRP family and possesses activity related to the Wntsignal pathway.

65. Full-Length PRO717 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO717. In particular, Applicants have identified and isolated cDNAencoding a PRO717 polypeptide, as disclosed in further detail in theExamples below. To Applicants present knowledge, the UNQ385(DNA50988-1326) nucleotide sequence encodes a novel factor; using BLASTand FastA sequence alignment computer programs, no significant sequenceidentities to any known human proteins were revealed.

66. Full-Length PRO731 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO731. In particular, Applicants have identified and isolated cDNAencoding a PRO731 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various positions of the PRO731polypeptide have significant homology with the protocadherins 4, 68, 43,42, 3, and 5. Accordingly, it is presently believed that PRO731polypeptide disclosed in the present application is a newly identifiedmember of the protocadherin family and possesses cell-cell aggregationor signaling activity or signal transduction involvement typical of thisfamily.

67. Full-length PRO218 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO218. In particular, Applicants have identified and isolated cDNAencoding a PRO218 polypeptide, as disclosed in further detail in theExamples below. The PRO218-encoding clone was isolated from a humanfetal kidney library. To Applicants present knowledge, the UNQ192(DNA30867-1335) nucleotide sequence encodes a novel factor; using BLASTand FastA sequence alignment computer programs, no significant sequenceidentities to any known proteins were revealed. Some sequence identitywas found with membrane regulator proteins, indicating that PRO218 mayfunction as a membrane regulator.

68. Full-Length PRO768 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO768. In particular, Applicants have identified and isolated cDNAencoding a PRO768 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO768polypeptide have significant homology with integrins, including integrin7 and 6. Accordingly, it is presently believed that PRO768 polypeptidedisclosed in the present application is a newly identified member of theintegrin family, either a homologue or a splice variant of integrin 7,and is involved with cell adhesion and communication between musclecells and the extracellular matrix.

69. Full-Length PRO771 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO771. In particular, Applicants have identified and isolated cDNAencoding a PRO771 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO771polypeptide have significant sequence identity and similarity withtestican. Accordingly, it is presently believed that PRO771 polypeptidedisclosed in the present application is a newly identified member of thetestican family and possesses cell signaling, binding, or adhesionproperties, typical of this family.

70. Full-Length PRO733 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO733. In particular, Applicants have identified and isolated cDNAencoding a PRO733 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO733polypeptide nave significant sequence identity with the T1/ST receptorbinding protein. Accordingly, it is presently believed that PRO733polypeptide disclosed in the present application is a newly identifiedmember of the interleulin-like family binding proteins which may be acytokine and which may be involved in cell signaling. It is believedthat PRO733 is an ApoAIV homologue.

71. Full-Length PRO162 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO162. In particular, Applicants have identified and isolated cDNAencoding a PRO162 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO162polypeptide have significant homology with human pancreatitis-associatedprotein (PAP). Applicants have also found that the DNA encoding thePRO162 polypeptide has significant homology with bovine lithostathineprecursor and bovine pancreatic thread protein (PTP). Accordingly, it ispresently believed that PRO162 polypeptide disclosed in the presentapplication is a newly identified member of the pancreatitis-associatedprotein family and possesses activity typical of thepancreatitis-associated protein family.

72. Full-Length PRO788 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO788. In particular, Applicants have identified and isolated cDNAencoding a PRO788 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO788polypeptide have significant homology with the anti-neoplastic urinaryprotein. Applicants have also found that the DNA encoding the PRO788polypeptide has significant homology with human E48 antigen, humancomponent B protein, and human prostate stem cell antigen. Accordingly,it is presently believed that PRO788 polypeptide disclosed in thepresent application is a newly identified member of the anti-neoplasticurinary protein family and possesses anti-neoplastic activity typical ofthe anti-neoplastic urinary protein family.

73. Full-Length PRO1008 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1008. In particular, Applicants have identified and isolated cDNAencoding a PRO1008 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO1008polypeptide have significant sequence identity and similarity with mousedkk-1 (mdkk-1). Accordingly, it is presently believed that PRO1008polypeptide disclosed in the present application is a newly identifiedmember of the dkk-1 family and possesses head inducing activity typicalof this family.

74. Full-Length PRO1012 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1012. In particular, Applicants have identified and isolated cDNAencoding a PRO1012 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO1012polypeptide have sequence identity with disulfide isomerase.Accordingly, it is presently believed that PRO1012 polypeptide disclosedin the present application is a newly identified member of the ERretained protein family and possesses activity related to theprocessing, production and/or folding of polypeptides typical of thedisulfide isomerase family.

75. Full-Length PRO1014 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1014. In particular, Applicants have identified and isolated cDNAencoding a PRO1014 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO1014polypeptide have sequence identity with reductase and dehydrogenase.Accordingly, it is presently believed that PRO1014 polypeptide disclosedin the present application is a newly identified member of the reductasesuper family and possesses reduction capabilities typical of thisfamily.

76. Full-Length PRO1017 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1017. In particular, Applicants have identified and isolated cDNAencoding a PRO1017 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO1017polypeptide have sequence identity with HNK-1 sulfotransferase.Accordingly, it is presently believed that PRO1017 polypeptide disclosedin the present application is a newly identified member of the HNK-1sulfotransferase family and is involved with the synthesis of HNK-1carbohydrate epitopes typical of this family.

77. Full-Length PRO474 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO474. In particular, Applicants have identified and isolated cDNAencoding a PRO474 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO474polypeptide have sequence identity with dehydrogenase, glucosedehydrogenase and oxidoreductase. Accordingly, it is presently believedthat PRO474 polypeptide disclosed in the present application is a newlyidentified member of the dehydrogenase family and is involved in theoxidation of glucose.

78. Full-length PRO1031 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1031. In particular, Applicants have identified and isolated cDNAencoding a PRO1031 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO1031polypeptide have sequence identity with IL-17 and CTLA-8. Accordingly,it is presently believed that PRO1031 polypeptide disclosed in thepresent application is a newly identified member of the cytokine familyand thus may be involved in inflammation and/or the immune system.

79. Full-Length PRO938 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO938. In particular, Applicants have identified and isolated cDNAencoding a PRO938 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO938 polypeptide has significantsimilarity to protein disulfide isomerase. Accordingly, it is presentlybelieved that PRO938 polypeptide disclosed in the present application isa newly identified member of the thioredoxin family proteins andpossesses activity typical of protein disulfide isomerase.

80. Full-Length PRO1082 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1082. In particular, Applicants have identified and isolated cDNAencoding a PRO1082 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO1082polypeptide have sequence identity with a lectin-like oxidized LDLreceptor appearing in the database as “AB010710_(—)1”. Accordingly, itis presently believed that PRO1082 polypeptide disclosed in the presentapplication is a newly identified member of the LDL receptor family.

81. Full-Length PRO1083 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1083. In particular, Applicants have identified and isolated cDNAencoding a PRO1083 polypeptide, as disclosed in further detail in theExamples below. The PRO1083-encoding clone was isolated from a humanfetal kidney library using a trapping technique which selects fornucleotide sequences encoding secreted proteins. To Applicants presentknowledge, the UNQ540 (DNA50921-1458) nucleotide sequence encodes anovel factor; using BLAST and FastA sequence alignment computerprograms, some sequence identity with a 7TM receptor, latrophilinrelated protein 1 and a macrophage restricted cell surface glycoproteinwas shown. The kinase phosphorylation site and G-coupled receptor domainshown in FIG. 204 indicate that PRO1083 is a novel member of the 7TMreceptor superfamily.

82. Full-Length PRO200 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas VEGF-E. In particular, Applicants have identified and isolated cDNAencoding a VEGF-E polypeptide, as disclosed in further detail in theExamples below. Using BLAST sequence alignment computer programs,Applicants found that the VEGF-E polypeptide has significant homologywith VEGF and bone morphogenetic protein 1. In particular, the cDNAsequence of VEGF-E exhibits 24% amino acid similarity with VEGF and hasstructural conservation. In addition, VEGF-E contains a N-terminal halfwhich is not present in VEGF and that has 28% homology to bonemorphogenetic protein 1.

83. Full-Length PRO285 and PRO286 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO285 and PRO286 In particular, Applicants have identified andisolated cDNAs encoding PRO285 and PRO286 polypeptides, as disclosed infurther detail in the Examples below. Using BLAST and FastA sequencealignment computer programs, Applicants found that the coding sequencesof PRO285 and PRO286 are highly homologous to DNA sequencesHSU88540_(—)1, HSU88878_(—)1, HSU88879_(—)1, HSU88880_(—)1, andHSU88881_(—)1 in the GenBank database.

Accordingly, it is presently believed that the PRO285 and PRO286proteins disclosed in the present application are newly identified humanhomologues of the Drosophila protein Toll, and are likely to play animportant role in adaptive immunity. More specifically, PRO285 andPRO286 may be involved in inflammation, septic shock, and response topathogens, and play possible roles in diverse medical conditions thatare aggravated by immune response, such as, for example, diabetes, ALS,cancer, rheumatoid arthritis, and ulcers. The role of PRO285 and PRO286as pathogen pattern recognition receptors, sensing the presence ofconserved molecular structures present on microbes, is further supportedby the data disclosed in the present application, showing that a knownhuman Toll-like receptor, TLR2 is a direct mediator of LPS signaling.

84. Full-Length PRO213-1. PRO1330 and PRO1449 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO213-1, PRO1330 and/or PRO1449. In particular, cDNA encoding aPRO213-1, PRO1330 and/or PRO1449 polypeptide has been identified andisolated, as disclosed in further detail in the Examples below. It isnoted that proteins produced in separate expression rounds may be givendifferent PRO numbers but the UNQ number is unique for any given DNA andthe encoded protein, and will not be changed. However, for sake ofsimplicity, in the present specification the protein encoded byDNA30943-1163-1, DNA64907-1163-1 and DNA64908-1163-1 as well as allfurther native homologues and variants included in the foregoingdefinition of PRO213-1, PRO1330 and/or PRO1449, will be referred to as“PRO213-1, PRO1330 and/or PRO1449”, regardless of their origin or modeof preparation.

85. Full-Length PRO298 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO298. (It is noted that PRO298 is an arbitrary designation of aprotein encoded by the nucleic acid shown in FIG. 218, SEQ ID NO:514,and having the amino acid sequence shown in FIG. 219, SEQ ID NO:515.Further proteins having the same amino acid sequence but expressed indifferent rounds of expression, may be given different “PRO” numbers.)

In particular, Applicants have identified and isolated cDNA encoding aPRO298 polypeptide, as disclosed in further detail in the Examplesbelow. Using BLASTX 2.0a8MP-WashU computer program, socring parameters:T=12, S=68, S2=36, Matrix: BLOSUM62, Applicants found that the PRO298protein specifically disclosed herein shows a limited (27–38%) sequenceidentity with the following sequences found in the GenBank database:S59392 (probable membrane protein YLR246w—yeast); S58154 hypotheticalprotein SPAC2F7.10—yeast); CELF33D11_(—)9 (F33D11.9b—Caenorhabditiselegans); YO41_CAEEL (hypothetical 68.7 kd protein zk757.1); CEAC3_(—)5(AC3.4—Caenorhabditis elegans); S52691 (probable transmembrane proteinYDR126w—yeast); ATT12H17_(—)14 (protein—Arabidopsis thaliana); S55963(probable membrane protein YNL326c—yeast); CELC43H6_(—)2(C43H6.7—Caenorhabditis elegans); TMO18A10_(—)14 (A_TMO18A10.8—Arabinosathaliana).

86. Full-Length PRO337 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO337. In particular, Applicants have identified and isolated cDNAencoding a PRO337 polypeptide, as disclosed in further detail in theExamples below. Using BLAST, BLAST-2 and FastA sequence alignmentcomputer programs, Applicants found that a full-length native sequencePRO337 has 97% amino acid sequence identity with rat neurotrimin, 85%sequence identity with chicken CEPU, 73% sequence identity with chickenG55, 59% homology with human LAMP and 84% homology with human OPCAM.Accordingly, it is presently believed that PRO337 disclosed in thepresent application is a newly identified member of the IgLON sub familyof the immunoglobulin superfamily and may possess neurite growth anddifferentiation potentiating properties.

87. Full-Length PRO403 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO403. In particular, Applicants have identified and isolated cDNAencoding a PRO403 polypeptide, as disclosed in further detail in theExamples below. Using a BLAST, BLAST-2 and FastA sequence alignmentcomputer programs, Applicants found that a full-length native sequencePRO403 has 94% identity to bovine ECE-2 and 64% identity to human ECE-1.Accordingly is presently believed that PRO403 is a new member of the ECEprotein family and may posses ability to catalyze the production ofactive endothelin.

B. PRO Polypeptide Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence PRO or in various domainsof the PRO described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, gIu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081–1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO

Covalent modifications of PRO are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the PRO. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO to a water-insolublesupport matrix or surface for use in the method for purifying anti-PROantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79–86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259–306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO comprises linking the PROpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The PRO of the present invention may also be modified in a way to form achimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl- terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159–2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610–3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547–553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 6:1204–1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192–194 (1992)]; an α-tubulinepitope peptide [Skinner et al., J. Biol. Chem., 266:15163–15166(1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA, 87:6393–6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of PRO

The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149–2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

1. Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared fromtissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO oroligonucleotides of at least about 20–80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456–457(1978) can be employed. General aspects of mammal cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527–537 (1990) and Mansour et al.,Nature, 336:348–352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coil X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO-encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 [1981]; EP 139,383 published May 2, 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975(1991)) such as, e.g., K. lactis (MW98–8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737–742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265–278[i988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259–5263 [1979]);Schwannionyces such as Schwanniomyces occidentals (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284–289 [1983]; Tilburn et al., Gene,26:205–221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470–1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475–479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243–251 (1980)); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT) 060562,ATCC CCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PROencoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21–25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100–270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620–625 (1981); Mantei et al.,Nature, 281:40–46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201–5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

E. Uses for PRO

Nucleotide sequences (or their complement) encoding PRO have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

The full-Length native sequence PRO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or 35S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO nucleic acids include antisense orsense oligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRODNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO coding sequences.

Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO can be used to construct aPRO “knock out” animal which has a defective or altered gene encodingPRO as a result of homologous recombination between the endogenous geneencoding PRO and altered genomic DNA encoding PRO introduced into anembryonic stem cell of the animal. For example, cDNA encoding PRO can beused to clone genomic DNA encoding PRO in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capeechi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113–152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the PROpolypeptide.

Nucleic acid encoding the PRO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143–4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205–210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429–4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410–3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808–813 (1992).

The PRO polypeptides described herein may also be employed as molecularweight markers for protein electrophoresis purposes and the isolatednucleic acid sequences may be used for recombinantly expressing thosemarkers.

The nucleic acid molecules encoding the PRO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each PRO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The PRO polypeptides and nucleic acid molecules of the present inventionmay also be used for tissue typing, wherein the PRO polypeptides of thepresent invention may be differentially expressed in one tissue ascompared to another. PRO nucleic acid molecules will find use forgenerating probes for PCR, Northern analysis, Southern analysis andWestern analysis.

The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42–96.

When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795–799 (1996); Yasuda, Biomed. Ther., 27:1221–1223 (1993); Horaet al., Bio/Technology, 8:755–758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439–462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1–41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245–246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578–9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789–5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro- sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO polypeptide, thereby blocking the normalbiological activity of the PRO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For fer details see, e.g., Rossi, Current Biology,4:469–471 (1994), and PCT publication No. WO 97/33551 (published Sep.18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For farther detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

PRO213 polypeptides and portions thereof which possess the ability toregulate the growth induction cascade and/or the blood coagulationcascade may also be employed for such purposes both in vivo therapy andin vitro. Those of ordinary skill in the art will well know how toemploy PRO213 polypeptides for such uses.

PRO274 polypeptides and portions thereof which have homology to 7TMprotein and Fn54 may also be useful for in vivo therapeutic purposes, aswell as for various other applications. The identification of novel 7TMprotein and Fn54-like molecules may have relevance to a number of humandisorders which involve recognition of ligands and the subsequent signaltransduction of information contained within those ligands in order tocontrol cellular processes. Thus, the identification of new 7TM proteinand Fn54-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as in various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO274.

PRO300 polypeptides and portions thereof which have homology to Diff 33may also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel Diff 33-likemolecules may have relevance to a number of human disorders such as thephysiology of cancer. Thus, the identification of new Diff 33-likemolecules is of special importance in that such proteins may serve aspotential therapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as various industrial applications. As aresult, there is particular scientific and medical interest in newmolecules, such as PRO300.

PRO296 polypeptides of the present invention which possess biologicalactivity related to that of the sarcoma-amplified SAS protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO296polypeptides of the present invention for such purposes.

PRO329 polypeptides of the present invention which possess biologicalactivity related to that of immunoglobulin F_(c) receptor protein orsubunit thereof may be employed both in vivo for therapeutic purposesand in vitro. Those of ordinary skill in the art will well know how toemploy the PRO329 polypeptides of the present invention for suchpurposes.

PRO362 polypeptides of the present invention which possess biologicalactivity related to that of the A33 antigen protein, HCAR protein or theNrCAM related cell adhesion molecule may be employed both in vivo fortherapeutic purposes and in vitro.

PRO363 polypeptides of the present invention which possess biologicalactivity related to that of the cell surface HCAR protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO363polypeptides of the present invention for such purposes. Specifically,extracellular domains derived from the PRO363 polypeptides may beemployed therapeutically in vivo for lessening the effects of viralinfection.

PRO868 polypeptides of the present invention which possess biologicalactivity related to that of the tumor necrosis factor protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO868polypeptides of the present invention for such purposes.

PRO382 polypeptides of the present invention which possess biologicalactivity related to that of the serine protease proteins may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO382 polypeptides ofthe present invention for such purposes.

PRO545 polypeptides and portions thereof which have homology to meltrinmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel moleculesassociated with cellular adhesion may be relevant to a number of humandisorders. Given that the meltrin proteins may play an important role ina number of disease processes, the identification of new meltrinproteins and meltrin-like molecules is of special importance in thatsuch proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research, as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO545.

PRO617 polypeptides of the present invention which possess biologicalactivity related to that of the CD24 protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO617 polypeptides of thepresent invention for such purposes.

PRO700 polypeptides and portions thereof which have homology to proteindisulfide isomerase may also be useful for in vivo therapeutic purposes,as well as for various other applications. The identification of novelprotein disulfide isomerases and related molecules may be relevant to anumber of human disorders. Given that formation of disulfide bonds andprotein folding play important roles in a number of biologicalprocesses, the identification of new protein disulfide isomerases andprotein disulfide isomerase-like molecules is of special importance inthat such proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research, as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO700.

PRO702 polypeptides of the present invention which possess biologicalactivity related to that of the conglutinin protein may be employed bothin vivo for therapeutic purposes and in vitro. Those of ordinary skillin the art will well know how to employ the PRO702 polypeptides of thepresent invention for such purposes. PRO702 polypeptides havingconglutinin activity would be expected to be capable of inhibitinghaemagglutinin activity by influenza viruses and/or function asimmunoglobulin-independent defense molecules as a result of acomplement-mediated mechanism.

PRO703 polypeptides of the present invention which possess biologicalactivity related to that of the VLCAS protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO703 polypeptides of thepresent invention for such purposes.

PRO703 polypeptides and portions thereof which have homology to VLCASmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel VLCAS proteinsand related molecules may be relevant to a number of human disorders.Thus, the identification of new VLCAS proteins and VLCAS protein-likemolecules is of special importance in that such proteins may serve aspotential therapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as various industrial applications. As aresult, there is particular scientific and medical interest in newmolecules, such as PRO703.

PRO705 polypeptides of the present invention which possess biologicalactivity related to that of the K-glypican protein may be employed bothin vivo for therapeutic purposes and in vitro. Those of ordinary skillin the art will well know how to employ the PRO705 polypeptides of thepresent invention for such purposes.

PRO708 polypeptides of the present invention which possess biologicalactivity related to that of the aryl sulfatase proteins may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO708 polypeptides ofthe present invention for such purposes.

PRO320 polypeptides of the present invention which possess biologicalactivity related to that of the fibulin protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO320 polypeptides of thepresent invention for such purposes.

PRO320 polypeptides and portions thereof which have homology to fibulinmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel fibulin proteinsand related molecules may be relevant to a number of human disorderssuch as cancer or those involving connective tissue, attachmentmolecules and related mechanisms. Thus, the identification of newfibulin proteins and fibulin protein-like molecules is of specialimportance in that such proteins may serve as potential therapeutics fora variety of different human disorders. Such polypeptides may also playimportant roles in biotechnological and medical research as well asvarious industrial applications. As a result, there is particularscientific and medical interest in new molecules, such as PRO320.

PRO324 polypeptides of the present invention which possess biologicalactivity related to that of oxidoreductases may be employed both in vivofor therapeutic purposes and in vitro. Those of ordinary skill in theart will well know how to employ the PRO324 polypeptides of the presentinvention for such purposes.

PRO351 polypeptides of the present invention which possess biologicalactivity related to that of the prostasin protein may be employed bothin vivo for therapeutic purposes and in vitro. Those of ordinary skillin the art will well know how to employ the PRO351 polypeptides of thepresent invention for such purposes.

PRO351 polypeptides and portions thereof which have homology toprostasin may also be useful for in vivo therapeutic purposes, as wellas for various other applications. The identification of novel prostasinproteins and related molecules may be relevant to a number of humandisorders. Thus, the identification of new prostasin proteins andprostasin-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO351.

PRO352 polypeptides of the present invention which possess biologicalactivity related to that of the butyrophilin protein may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO352 polypeptides ofthe present invention for such purposes.

PRO381 polypeptides of the present invention which possess biologicalactivity related to that of one or more of the FKPB immunophilinproteins may be employed both in vivo for therapeutic purposes and invitro, for example for enhancing immunosuppressant activity and/or foraxonal regeneration. Those of ordinary skill in the art will well knowhow to employ the PRO381 polypeptides of the present invention for suchpurposes.

PRO386 polypeptides of the present invention which possess biologicalactivity related to that of the beta-2 subunit of a sodium channelexpressed in mammalian cells may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO386 polypeptides of the presentinvention for such purposes.

PRO540 polypeptides of the present invention which possess biologicalactivity related to that of the LCAT protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO540 polypeptides of thepresent invention for such purposes.

PRO615 polypeptides of the present invention which possess biologicalactivity related to that of the synaptogyrin protein may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO615 polypeptides ofthe present invention for such purposes.

PRO615 polypeptides and portions thereof which have homology tosynaptogyrin may also be useful for in vivo therapeutic purposes, aswell as for various other applications. The identification of novelsynaptogyrin proteins and related molecules may be relevant to a numberof human disorders. Thus, the identification of new synaptogyrinproteins and synaptogyrin-like molecules is of special importance inthat such proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research as well as variousindustrial applications. As a result, there is particular scientifican(d medical interest in new molecules, such as PRO615.

PRO618 polypeptides of the present invention which possess biologicalactivity related to that of an enteropeptidase may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO618 polypeptides of thepresent invention for such purposes.

PRO618 polypeptides and portions thereof which have homology toenteropeptidase may also be useful for in vivo therapeutic purposes, aswell as for various other applications. The identification of novelenteropeptidase proteins and related molecules may be relevant to anumber of human disorders. Thus, the identification of newenteropeptidase proteins and enteropeptidase-like molecules is ofspecial importance in that such proteins may serve as potentialtherapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as various industrial applications. As aresult, there is particular scientific and medical interest in newmolecules, such as PRO618.

PRO719 polypeptides of the present invention which possess biologicalactivity related to that of the lipoprotein lipase H protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO719polypeptides of the present invention for such purposes.

PRO724 polypeptides of the present invention which possess biologicalactivity related to that of the human LDL receptor protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO724polypeptides of the present invention for such purposes.

PRO772 polypeptides of the present invention which possess biologicalactivity related to that of the human A4 protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO772 polypeptides of thepresent invention for such purposes.

PRO852 polypeptides of the present invention which possess biologicalactivity related to that of certain protease protein may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO852 polypeptides ofthe present invention for such purposes.

PRO853 polypeptides of the present invention which possess biologicalactivity related to that of the reductase protein may be employed bothin vivo for therapeutic purposes and in vitro. Those of ordinary skillin the art will well know how to employ the PRO853 polypeptides of thepresent invention for such purposes.

PRO853 polypeptides and portions thereof which have homology toreductase proteins may also be useful for in vivo therapeutic purposes,as well as for various other applications. Given that oxygen freeradicals and antioxidants appear to play important roles in a number ofdisease processes, the identification of new reductase proteins andreductase-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO853.

PRO860 polypeptides of the present invention which possess biologicalactivity related to that of the neurofascin protein may be employed bothin vivo for therapeutic purposes and in vitro. Those of ordinary skillin the art will well know how to employ the PRO860 polypeptides of thepresent invention for such purposes.

PRO860 polypeptides and portions thereof which have homology toneurofascin may also be useful for in vivo therapeutic purposes, as wellas for various other applications. The identification of novelneurofascin proteins and related molecules may be relevant to a numberof human disorders which involve cellular adhesion. Thus, theidentification of new neurofascin proteins and neurofascin protein-likemolecules is of special importance in that such proteins may serve aspotential therapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as various industrial applications. As aresult, there is particular scientific and medical interest in newmolecules, such as PRO860.

PRO846 polypeptides of the present invention which possess biologicalactivity related to that of the CMRF35 protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO846 polypeptides of thepresent invention for such purposes.

PRO846 polypeptides and portions thereof which have homology to theCMRF35 protein may also be useful for in vivo therapeutic purposes, aswell as for various other applications. The identification of novelCMRF35 protein and related molecules may be relevant to a number ofhuman disorders. Thus, the identification of new CMRF35 protein andCMRF35 protein-like molecules is of special importance in that suchproteins may serve as potential therapeutics for a variety of differenthuman disorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO846.

PRO862 polypeptides of the present invention which possess biologicalactivity related to that of the lysozyme protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO862 polypeptides of thepresent invention for such purposes.

PRO862 polypeptides and portions thereof which have homology to thelysozyme protein may also be useful for in vivo therapeutic purposes, aswell as for various other applications. The identification of novellysozyme proteins and related molecules may be relevant to a number ofhuman disorders. Thus, the identification of new lysozymes andlysozyme-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO862.

PRO864 polypeptides of the present invention which possess biologicalactivity related to that of the Wnt-4 protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO864 polypeptides of thepresent invention for such purposes.

PRO864 polypeptides and portions thereof which have homology to theWnt-4 protein may also be useful for in vivo therapeutic purposes, aswell as for various other applications. The identification of novelWnt-4 proteins and related molecules may be relevant to a number ofhuman disorders. Thus, the identification of new Wnt-4 protein and Wnt-4protein-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO864.

PRO792 polypeptides of the present invention which possess biologicalactivity related to that of the CD23 protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO792 polypeptides of thepresent invention for such purposes.

PRO866 polypeptides of the present invention which possess biologicalactivity related to that of mindin and/or spondin protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO866polypeptides of the present invention for such purposes.

PRO871 polypeptides of the present invention which possess biologicalactivity related to that of the cyclophilin protein family may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO871polypeptides of the present invention for such purposes.

PRO873 polypeptides of the present invention which possess biologicalactivity related to that of carboxylesterases may be employed both invivo for therapeutic purposes and in vitro. For example, they be used inconjunction with prodrugs to convert the prodrug to its active form (seeDanks et al., supra). They may be used to inhibit parasite infection(see van Pelt et al., supra). Methods for employ the PRO873 polypeptidesof the present invention for these, and other purposes will be readilyapparent to those of ordinary skill in the art.

PRO940 polypeptides of the present invention which possess biologicalactivity related to that of the CD33 protein and/or OB binding protein-2may be employed both in vivo for therapeutic purposes and in vitro.Those of ordinary skill in the art will well know how to employ thePRO940 polypeptides of the present invention for such purposes.

PRO941 polypeptides of the present invention which possess biologicalactivity related to that of a cadherin protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO941 polypeptides of thepresent invention for such purposes.

PRO944 polypeptides of the present invention which possess biologicalactivity related to that of the CPE-R protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO944 polypeptides of thepresent invention for such purposes. PRO944 polypeptides of the presentinvention that function to bind to Clostridium perfringens enterotoxin(CPE) may find use for effectively treating infection by the CPEendotoxin.

PRO983 polypeptides of the present invention which possess biologicalactivity related to that of the vesicle-associated membrane protein,VAP-33, may be employed both in vivo for therapeutic purposes and invitro. Those of ordinary skill in the art will well know how to employthe PRO983 polypeptides of the present invention for such purposes.

PRO1057 polypeptides of the present invention which possess biologicalactivity related to that of protease proteins may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art win well know how to employ the PRO1057 polypeptides of thepresent invention for such purposes.

PRO1071 polypeptides of the present invention which possess biologicalactivity related to that of the thrombospondin protein may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO1071 polypeptidesof the present invention for such purposes.

PRO1072 polypeptides of the present invention which possess biologicalactivity related to that of reductase proteins may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO1072 polypeptides of thepresent invention for such purposes.

PRO1075 polypeptides of the present invention which possess biologicalactivity related to that of protein disulfide isomerase may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO1075 polypeptidesof the present invention for such purposes.

PRO181 polypeptides of the present invention which possess biologicalactivity related to that of the cornichon protein may be employed bothin vivo for therapeutic purposes and in vitro. Those of ordinary skillin the art will well know how to employ the PRO181 polypeptides of thepresent invention for such purposes.

PRO827 polypeptides of the present invention which possess biologicalactivity related to that of various integrin proteins may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO827 polypeptides ofthe present invention for such purposes.

PRO1114 polypeptides of the present invention which possess biologicalactivity related to that of the cytokine receptor family of proteins maybe employed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO1114polypeptides of the present invention for such purposes.

In addition to the above, the PRO1114 interferon receptor polypeptidesmay be employed in applications, both in vivo and in vitro, where theability to bind to an interferon ligand is desired. Such applicationswill be well within the skill level in the art.

PRO237 polypeptides of the present invention which possess biologicalactivity related to that of the carbonic anhydrase protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO237polypeptides of the present invention for such purposes.

PRO541 polypeptides of the present invention which possess biologicalactivity related to that of a trypsin inhibitor protein may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO541 polypeptides ofthe present invention for such purposes.

PRO273 polypeptides can be used in assays that other chemokines would beused in to perform comparative assays. The results can be usedaccordingly.

PRO701 polypeptides of the present invention which possess biologicalactivity related to that of the neuroligin family may be employed bothin vivo for therapeutic purposes and in vitro. Those of ordinary skillin the art will well know how to employ the PRO701 polypeptides of thepresent invention for such purposes.

PRO701 can be used in assays with neurons and its activity thereon canbe compared with that of neuroligins 1, 2 and 3. The results can beapplied accordingly.

PRO704 polypeptides of the present invention which possess biologicalactivity related to that of vesicular integral membrane proteins may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO704polypeptides of the present invention for such purposes.

PRO704 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly. PRO704 can be tagged or measured for activity tomeasure endocytosis activity and thereby used to screen for agents whicheffect endocytosis.

PRO706 polypeptides of the present invention which possess biologicalactivity related to that of the endogenous prostatic acid phosphataseprecursor may be employed both in vivo for therapeutic purposes and invitro. Those of ordinary skill in the art will well know how to employthe PRO706 polypeptides of the present invention for such purposes.

PRO706 can be used in assays with human prostatic acid phosphatase orhuman lysosomal acid phosphatase and its activity thereon can becompared with that of human prostatic acid phosphatase or humanlysosomal acid phosphatase. The results can be applied accordingly.

PRO707 polypeptides of the present invention which possess biologicalactivity related to that of cadherins may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO707 polypeptides of the presentinvention for such purposes.

PRO707 can be used in assays to determine its activity in relation toother cadherins, particularly cadherin FIB3. The results can be appliedaccordingly.

PRO322 polypeptides of the present invention which possess biologicalactivity related to that of neuropsin may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO322 polypeptides of the presentinvention for such purposes.

PRO322 can be used in assays to determine its activity relative toneuropsin, trypsinogen, serine protease and neurosin, and the resultsapplied accordingly.

PRO526 polypeptides of the present invention which possess biologicalactivity related to that of protein-protein binding proteins may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO526polypeptides of the present invention for such purposes.

Assays can be performed with growth factors and other proteins which areknown to form complexes to determine whether PRO526 binds thereto andwhether there is increased half-life due to such binding. The resultscan be used accordingly.

PRO531 polypeptides of the present invention which possess biologicalactivity related to that of the protocadherins may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO531 polypeptides of thepresent invention for such purposes.

PRO531 can be used in assays against protocadherin 3 and otherprotocadherins, to determine their relative activities. The results canbe applied accordingly.

PRO534 polypeptides of the present invention which possess biologicalactivity related to that of the protein disulfide isomerase may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO534polypeptides of the present invention for such purposes.

PRO534 can be used in assays with protein disulfide isomerase todetermine the relative activities. The results can be appliedaccordingly.

PRO697 polypeptides of the present invention which possess biologicalactivity related to that of the sFRP family may be employed both in vivofor therapeutic purposes and in vitro. Those of ordinary skill in theart will well know how to employ the PRO697 polypeptides of the presentinvention for such purposes.

PRO697 can be used in assays with sFRPs and SARPs to determine therelative activities. The results can be applied accordingly.

PRO731 polypeptides of the present invention which possess biologicalactivity related to that of any protocadherin may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO731 polypeptides of thepresent invention for such purposes.

PRO731 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO768 polypeptides of the present invention which possess biologicalactivity related to that of integrins may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO768 polypeptides of the presentinvention for such purposes.

PRO768 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO771 polypeptides of the present invention which possess biologicalactivity related to that of the testican protein may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO771 polypeptides of thepresent invention for such purposes.

PRO771 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO733 polypeptides of the present invention which possess biologicalactivity related to that of the proteins which bind the T1/ST2 receptormay be employed both in vivo for therapeutic purposes and in vitro.Those of ordinary skill in the art will well know how to employ thePRO733 polypeptides of the present invention for such purposes.

PRO733 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO162 polypeptides of the present invention which possess biologicalactivity related to that of the pancreatitis-associated protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO162polypeptides of the present invention for such purposes.

PRO162 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO788 polypeptides of the present invention which possess biologicalactivity related to that of the anti-neoplastic urinary protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO788polypeptides of the present invention for such purposes.

PRO788 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO1008 polypeptides of the present invention which possess biologicalactivity related to that of dkk-1 may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO1008 polypeptides of the presentinvention for such purposes.

PRO1008 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO1012 polypeptides of the present invention which possess biologicalactivity related to that of the protein disulfide isomerase may beemployed both in vivo and in vitro purposes. Those of ordinary skill inthe art will well know how to employ the PRO1012 polypeptides of thepresent invention for such purposes.

PRO1012 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO1014 polypeptides of the present invention which possess biologicalactivity related to that of reductase may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO1014 polypeptides of the presentinvention for such purposes.

PRO1014 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. Inhibitors ofPRO1014 are particularly preferred. The results can be appliedaccordingly.

PRO1017 polypeptides of the present invention which possess biologicalactivity related to that of sulfotransferase may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO1017 polypeptides of thepresent invention for such purposes.

PRO1017 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO474 polypeptides of the present invention which possess biologicalactivity related to that of dehydrogenase may be employed both in vivofor therapeutic purposes and in vitro. Those of ordinary skid in the artwill well know how to employ the PRO474 polypeptides of the presentinvention for such purposes.

PRO474 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO1031 polypeptides of the present invention which possess biologicalactivity related to that of IL-17 may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO1031 polypeptides of the presentinvention for such purposes.

PRO1031 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly.

PRO938 polypeptides of the present invention which possess biologicalactivity related to that of protein disulfide isomerase may be employedboth in vivo for therapeutic purposes and in vitro. Those of ordinaryskill in the art will well know how to employ the PRO938 polypeptides ofthe present invention for such purposes.

PRO1082 polypeptides of the present invention which possess biologicalactivity related to that of the LDL receptor may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO1082 polypeptides of thepresent invention for such purposes.

PRO1082 can be used in assays with the polypeptides to which they haveidentity with to determine the relative activities. The results can beapplied accordingly. PRO1082 can also be used in assays to identifycandidate agents which modulate the receptors.

PRO1083 polypeptides of the present invention which possess biologicalactivity related to that of 7TM receptors may be employed both in vivofor therapeutic purposes and in vitro. Those of ordinary skill in theart will well know how to employ the PRO1083 polypeptides of the presentinvention for such purposes.

In particular PRO1083 can be used in assays to determine candidateagents which control or modulate PRO1083, i.e., have an effect on thereceptor.

The VEGF-E molecules herein have a number of therapeutic uses associatedwith survival, proliferation and/or differention of cells. Such usesinclude the treatment of umbilical vein endothelial cells, in view ofthe demonstrated ability of VEGF-E to increase survival of humanumbilical vein endothelial cells. Treatment may be needed if the veinwere subjected to traumata, or situations wherein artificial means areemployed to enhance the survival of the umbilical vein, for example,where it is weak, diseased, based on an artificial matrix, or in anartificial environment. Other physiological conditions that could beimproved based on the selective mitogenic character of VEGF-E are alsoincluded herein. Uses also include the treatment of fibroblasts andmyocytes, in view of the demonstrated ability of VEGF-E to induceproliferation of fibroblasts and hypertrophy in myocytes. In particular,VEGF-E can be used in wound healing, tissue growth and muscle generationand regeneration.

For the indications referred to above, the VEGF-E molecule will beformulated and dosed in a fashion consistent with good medical practicetaking into account the specific disorder to be treated, the conditionof the individual patient, the site of delivery of the VEGF-E, themethod of administration, and other factors known to practitioners.Thus, for purposes herein, the “therapeutically effective amount” of theVEGF-E is an amount that is effective either to prevent, lessen theworsening of, alleviate, or cure the treated condition, in particularthat amount which is sufficient to enhance the survival, proliferationand/or differentiation of the treated cells in vivo.

VEGF-E amino acid variant sequences and derivatives that areimmunologically crossreactive with antibodies raised against native VEGFare useful in immunoassays for VEGF-E as standards, or, when labeled, ascompetitive reagents.

The VEGF-E is prepared for storage or administration by mixing VEGF-Ehaving the desired degree of purity with physiologically acceptablecarriers, excipients, or stabilizers. Such materials are non-toxic torecipients at the dosages and concentrations employed. If the VEGF-E iswater soluble, it may be formulated in a buffer such as phosphate orother organic acid salt preferably at a pH of about 7 to 8. If theVEGF-E is only partially soluble in water, it may be prepared as amicroemulsion by formulating it with a nonionic surfactant such asTween, Pluronics, or PEG, e.g., Tween 80, in an amount of 0.04–0.05%(w/v), to increase its solubility.

Optionally other ingredients may be added such as antioxidants, e.g.,ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; and sugar alcohols such asmannitol or sorbitol.

The VEGF-E to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). The VEGF-E ordinarilywin be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of theVEGF-E preparations typically will be about from 6 to 8, although higheror lower pH values may also be appropriate in certain instances. It willbe understood that use of certain of the foregoing excipients, carriers,or stabilizers will result in the formation of salts of the VEGF-E.

If the VEGF-E is to be used parenterally, therapeutic compositionscontaining the VEGF-E generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Generally, where the disorder permits, one should formulate and dose theVEGF-E for site-specific delivery. This is convenient in the case ofwounds and ulcers. Sustained release formulations may also be prepared,and include the formation of microcapsular particles and implantablearticles. For preparing sustained-release VEGF-E compositions, theVEGF-E is preferably incorporated into a biodegradable matrix ormicrocapsule. A suitable material for this purpose is a polylactide,although other polymers of poly-(a-hydroxycarboxylic acids), such aspoly-D-(−)-3-hydroxybutyric acid (EP 133,988A), can be used. Otherbiodegradable polymers include poly(lactones), poly(acetals),poly(orthoesters), or poly(orthocarbonates). The initial considerationhere must be that the carrier itself, or its degradation products, isnontoxic in the target tissue and will not further aggravate thecondition. This can be determined by routine screening in animal modelsof the target disorder or, if such models are unavailable, in normalanimals. Numerous scientific publications document such animal models.

For examples of sustained release compositions, see U.S. Pat. No.3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, CanadianPatent No. 1176565, U. Sidman et al., Biopolymers 22, 547 [1983], and R.Langer et al., Chem. Tech. 12, 98 [1982].

When applied topically, the VEGF-E is suitably combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of such other ingredients, except that they must bepharmaceutically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

For obtaining a gel formulation, the VEGF-E formulated in a liquidcomposition may be mixed with an effective amount of a water-solublepolysaccharide or synthetic polymer such as polyethylene glycol to forma gel of the proper viscosity to be applied topically. Thepolysaccharide that may be used includes, for example, cellulosederivatives such as etherified cellulose derivatives, including alkylcelluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses,for example, methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose;starch and fractionated starch; agar; alginic acid and alginates; gumarabic; pullullan; agarose; carrageenan; dextrans; dextrans; fructans;inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans; andsynthetic biopolymers; as well as gums such as xanthan gum; guar gum;locust bean gum; gum arabic; tragacanth gum; and karaya gum; andderivatives and mixtures thereof. The preferred gelling agent herein isone that is inert to biological systems, nontoxic, simple to prepare,and not too runny or viscous, and will not destabilize the VEGF-E heldwithin it.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight polyethylene glycols to obtain the properviscosity. For example, a mixture of a polyethylene glycol of molecularweight 400–600 with one of molecular weight 1500 would be effective forthis purpose when mixed in the proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides andpolyethylene glycols is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient. Additionally, the cellulose derivatives may be in the formof alkali metal salts, for example, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2–5%, more preferably about 3%, of the gel and the VEGF is present in anamount of about 300–1000 mg per ml of gel.

The dosage to be employed is dependent upon the factors described above.As a general proposition, the VEGF-E is formulated and delivered to thetarget site or tissue at a dosage capable of establishing in the tissuea VEGF-E level greater than about 0.1 ng/cc up to a maximum dose that isefficacious but not unduly toxic. This intra-tissue concentration shouldbe maintained if possible by continuous infusion, sustained release,topical application, or injection at empirically determined frequencies.

It is within the scope hereof to combine the VEGF-E therapy with othernovel or conventional therapies (e.g., growth factors such as VEGF,aFGF, bFGF, PDGF, IGF, NGF, anabolic steroids, EGF or TGF-a) forenhancing the activity of any of the growth factors, including VEGF-E,in promoting cell proliferation, survival, differentiation and repair.It is not necessary that such cotreatment drugs be included per se inthe compositions of this invention, although this will be convenientwhere such drugs are proteinaceous. Such admixtures are suitablyadministered in the same manner and for the same purposes as the VEGF-Eused alone. The useful molar ratio of VEGF-E to such secondary growthfactors is typically 1:0.1–10, with about equimolar amounts beingpreferred.

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe PRO polypeptide hereof is combined in admixture with apharmaceutically acceptable carrier vehicle. Suitable carrier vehiclesand their formulation, inclusive of other human proteins, e.g., humanserum albumin, are described, for example, in Remington's PharmaceuticalSciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et aL thedisclosure of which is hereby incorporated by reference. The VEGF-Eherein may be administered parenterally to subjects suffering fromcardiovascular diseases or conditions, or by other methods that ensureits delivery to the bloodstream in an effective form.

Compositions particularly well suited for the clinical administration ofVEGF-E hereof employed in the practice of the present invention include,for example, sterile aqueous solutions, or sterile hydratable powderssuch as lyophiaed protein. It is generally desirable to include furtherin the formulation an appropriate amount of a pharmaceuticallyacceptable salt, generally in an amount sufficient to render theformulation isotonic. A pH regulator such as arginine base, andphosphoric acid, are also typically included in sufficient quantities tomaintain an appropriate pH, generally from 5.5 to 7.5. Moreover, forimprovement of shelf-life or stability of aqueous formulations, it mayalso be desirable to include further agents such as glycerol. hi thismanner, variant t-PA formulations are rendered appropriate forparenteral administration, and, in particular, intravenousadministration.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. For example, in the treatment of deep vein thrombosis orperipheral vascular disease, “bolus” doses, will typically be preferredwith subsequent administrations being given to maintain an approximatelyconstant blood level, preferably on the order of about 3 μg/ml.

However, for use in connection with emergency medical care facilitieswhere infusion capability is generally not available and due to thegenerally critical nature of the underlying disease (e.g., embolism,infarct), it will generally be desirable to provide somewhat largerinitial doses, such as an intravenous bolus.

For the various therapeutic indications referred to for the compoundshereof, the VEGF-E molecules will be formulated and dosed in a fashionconsistent with good medical practice taking into account the specificdisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners in the respective art. Thus, for purposes herein, the“therapeutically effective amount” of the VEGF-E molecules hereof is anamount that is effective either to prevent, lessen the worsening of,alleviate, or cure the treated condition, in particular that amountwhich is sufficient to enhance the survival, proliferation ordifferentiation of targeted cells in vivo. In general a dosage isemployed capable of establishing in the tissue that is the target forthe therapeutic indication being treated a level of a VEGF-E hereofgreater than about 0.1 ngcm³ up to a maximum dose that is efficaciousbut not unduly toxic. It is contemplated that intra-tissueadministration may be the choice for certain of the therapeuticindications for the compounds hereof.

The human Toll proteins of the present invention can also be used inassays to identify other proteins or molecules involved in Toll-mediatedsignal transduction. For example, PRO285 and PRO286 are useful inidentifying the as of yet unknown natural ligands of human Tolls, orother factors that participate (directly or indirectly) in theactivation of and/or signaling through a human Toll receptor, such aspotential Toll receptor associated kinases. In addition, inhibitors ofthe receptor/ligand binding interaction can be identified. Proteinsinvolved in such binding interactions can also be used to screen forpeptide or small molecule inhibitors or agonists of the bindinginteraction. Screening assays can be designed to find lead compoundsthat mimic the biological activity of a native Toll polypeptide or aligand for a native Toll polypeptide. Such screening assays will includeassays amenable to high-throughput screening of chemical libraries,making them particularly suitable for identifying small molecule drugcandidates. Small molecules contemplated include synthetic organic orinorganic compounds. The assays can be performed in a variety offormats, including protein-protein binding assays, biochemical screeningassays, immunoassays and cell based assays, which are well characterizedin the art.

In vitro assays employ a mixture of components including a Toll receptorpolypeptide, which may be part of fusion product with another peptide orpolypeptide, e.g., a tag for detecting or anchoring, etc. The assaymixtures may further comprise (for binding assays) a natural intra- orextracellular Toll binding target (i.e. a Toll ligand, or anothermolecule known to activate and/or signal through the Toll receptor).While native binding targets may be used, it is frequently preferred touse portion of such native binding targets (e.g. peptides), so long asthe portion provides binding affinity and avidity to the subject Tollprotein conveniently measurable in the assay. The assay mixture alsocontains a candidate pharmacological agent. Candidate agents encompassnumerous chemical classes, through typically they are organic compounds,preferably small organic compounds, and are obtained from a wide varietyof sources, including libraries of synthetic or natural compounds. Avariety of other reagents may also be included in the mixture, such as,salts, buffers, neutral proteins, e.g. albumin, detergents, proteaseinhibitors, nuclease inhibitors, antimicrobial agents, etc.

In in vitro binding assays, the resultant mixture is incubated underconditions whereby, but for the presence of the candidate molecule, theToll protein specifically binds the cellular binding target, portion oranalog, with a reference binding affinity. The mixture components can beadded in any order that provides for the requisite bindings andincubations may be performed at any temperature which facilitatesoptimal binding. Incubation periods are likewise selected for optimalbinding but also miniized to facilitate rapid high-throughput screening.

After incubation, the agent-biased binding between the Toll protein andone or more binding targets is detected by any convenient technique. Forcell-free binding type assays, a separation step is often used toseparate bound from unbound components. Separation may be effected byprecipitation (e.g. TCA precipitation, immunoprecipitation, etc.),immobilization (e.g. on a solid substrate), etc., followed by washingby, for example, membrane filtration (e.g. Whatman's P-18 ion exchangepaper, Polyfiltronic's hydrophobic GFC membrane, etc.), gelchromatography (e.g. gel filtration, affinity, etc.). For Toll-dependenttranscription assays, binding is detected by a change in the expressionof a Toll-dependent reporter.

Detection may be effected in any convenient way. For cell-free bindingassays, one of the components usually comprises or is coupled to alabel. The label may provide for direct detection as radioactivity,luminescence, optical or electron density, etc., or indirect detection,such as, an epitope tag, an enzyme, etc. A variety of methods may beused to detect the label depending on the nature of the label and otherassay components, e.g. through optical or electron density, radiativeemissions, nonradiative energy transfers, etc. or indirectly detectedwith antibody conjugates, etc.

Nucleic acid encoding the Toll polypeptides disclosed herein may also beused in gene therapy. In gene therapy applications, genes are introducedinto cells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both -conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene H therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83, 4143–4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205–210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429–4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410–3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256, 808–813 (1992).

The various uses listed in connection with the Toll proteins herein, arealso available for agonists of the native Toll receptors, which mimic atleast one biological function of a native Toll receptor.

Neurotrimin as well as other members of the IgLON subfamily of theimmunoglobulin superfamily have been identified to have effect uponneural patterning, differentiation, maturation and growth. As a result,PRO337 the human neurotrimin homolog polypeptides would be expected tohave utility in diseases which are characterized by neural disfunction.For example, motoneuron disorders such as amyotrophic lateral sclerosis(Lou Gehrig's disease), Bell's palsy, and various conditions involvingspinal muscular atrophy, or paralysis. NGF variant formulations of theinvention can be used to treat human neurodegenerative disorders, suchas Alzheimer's disease, Parkinson's disease, epilepsy, multiplesclerosis, Huntington's chorea, Down's Syndrome, nerve deafness, andMeniere's disease. Moreover PRO337 polypeptide may also be used as acognitive enhancer, to enhance learning particularly in dementia ortrauma, such as those associated with the above diseases.

Further, PRO337 may be employed to treat neuropathy, and especiallyperipheral neuropathy. “Peripheral neuropathy” refers to a disorderaffecting the peripheral nervous system, most often manifested as one ora combination of motor, sensory, sensorimotor, or autonomic neuraldysfunction. The wide variety of morphologies exhibited by peripheralneuropathies can each be attributed uniquely to an equally wide numberof causes. For example, peripheral neuropathies can be geneticallyacquired, can result from a systemic disease, or can be induced by atoxic agent. Examples include but are not limited to diabetic peripheralneuropathy, distal sensorimotor neuropathy, or autonomic neuropathiessuch as reduced motility of the gastrointestinal tract or atony of theurinary bladder. Examples of neuropathies associated with systemicdisease include post-polio syndrome or AIDS-associated neuropathy;examples of hereditary neuropathies include Charcot-Marie-Tooth disease,Refsum's disease, Abetalipoproteinemia, Tangier disease, Krabbe'sdisease, Metachromatic leukodystrophy, Fabry's disease, andDejerine-Sottas syndrome; and examples of neuropathies caused by a toxicagent include those caused by treatment with a chemotherapeutic agentsuch as vincristine, cisplatin, methotrexate, or3′-azido-3′-deoxythymidine. Correspondingly, neurotrimin antagonistswould be expected to have utility in diseases characterized by excessiveneuronal activity.

Endothelin is generated from inactive intermediates, the bigendothelins, by a unique processing event catalyzed by the zincmetalloprotease, endothelin converting enzyme (ECE). ECE was recentlycloned, and its structure was shown to be a single pass transmembraneprotein with a short intracellular N-terminal and a long extracellularC-terminal that contains the catalytic domain and numerousN-glycosylation sites. ECEs cleave the endothelin propeptide betweenTrp73 and Val74 producing the active peptide, ET, which appears tofunction as a local rather than a circulating hormone (Rubanyi, G. M. &Polokoff, M. A., Pharmachological Reviews 46: 325–415 (1994). Thus ECEactivity is a potential site of regulation of endothelin production anda possible target for therapeutic intervention in the endothelin system.By blocking ECE activity, it is possible stop the production of ET-1 byinhibiting the conversion of the relatively inactive precursor, bigET-1, to the physiologically active form.

ECE-2 is 64% identical to bovine ECE-2 at the amino acid level. ECE-2 isclosely related to ECE-1 (63% identical, 80% conserved), neutralendopeptidase 24.11 and the Kell blood group protein. Bovine ECE-2 is atype II membrane-bound metalloproteinase localized in the trans-Golginetwork where it acts as an intracellular enzyme converting endogenousbig endothelin-1 into active endothelin (Emoto, N. and Yanangisawa, M.,J. Biol. Chem. 270: 15262–15268 (1995). The bovine ECE-2 mRNA expressionis highest in parts of the brain, cerebral cortex, cerebellum andadrenal medulla. It is expressed at lower levels in mymetrium, testes,ovary, and endothelial cells. Bovine ECE-2 and ECE-1 both are moreactive on ET-1 as a substrate compared to ET-2 or ET-3, Emoto andYanangisawa, supra. Human ECE-2 is 736 amino acids in length with a 31residue amino-terminal tail, a 23 residue transmembrane helix and a 682carboxy-terminal domain. It is 94% identical to bovine ECE-2 and 64%identical to human ECE-1. The predicted transmembrane domain is highlyconserved between the human and bovine ECE-2 proteins and between humanECE-1 and human ECE-2, as are the putative N-linked glycosylation sites,Cys residues conserved in the neutral endopeptidase 24.11 and the Kellblood group protein family and the putative zinc binding motif. Thesequence suggests, that like other members of the NEP-ECE-Kell family,human ECE-2 encodes a type II transmembrane zinc-bindingmetalloproteinase, which, by extrapolation from what is known aboutbovine ECE-2, is an intracellular enzyme located within the secretorypathway which processes endogenously produced big ET-1 while it is stillin the secretory vesicles. Emoto and Yanangisawa, supra.

The expression pattern of ECE-2 differs from that observed for ECE-1.Northern blot analysis of mRNA levels indicated low levels of expressionof a 3.3 kb transcript in adult brain (highest in the cerebellum,putamen, medulla and temporal lobe, and lower in the cerebral cortex,occipital lobe and frontal lobe), spinal cord, lung and pancreas andhigher levels of a 4.5 kb transcript in fetal brain and kidney. The twotranscript sizes probably represent the use of alternativepolyadenylation sites as has been observed for bovine ECE-2 (Emoto andYanangisawa, supra) and ECE-1 (Xu et al., Cell 78: 473–485 (1994). PCRon cDNA libraries indicated low levels of expression in fetal brain,fetal kidney, fetal small intestine and adult testis. Fetal liver, fetallung and adult pancreas were all negative.

The endothelin (ET) family of peptides have potent vascular, cardiac andrenal actions which may be of pathophysiological importance in manyhuman disease states. ET-1 is expressed as an inactive 212 amino acidprepropeptide. The prepropeptide is first cleaved at Arg52-CysS3 andArg92-Ala93 and then the carboxy terminal Lys9l and Arg92 are trimmedfrom the protein to generate the propeptide big ET-1. ECEs then cleavethe propeptide between Trp73 and Val74, producing the active peptide,ET, which appears to function as a local rather than a circulatinghormone (Rubanyi and Polokoff, Pharma. R. 46: 325–415 (1994).

Endothelins may play roles in the pathophysiology of a number of diseasestates including: 1) cardiovascular diseases (vasospasm, hypertension,myocardial ischemia; reperfusion injury and acute myochardialinfarction, stroke (cerebral ischemia), congestive heart failure, shock,atherosclerosis, vascular thickening); 2) kidney disease (acute andchronic renal failure, glomerulonephritis, cirrhosis); 3) lung disease(bronchial asthma, pulmonary hypertension); 4) gastrointestinaldisorders (gastric ulcer, inflammatory bowel diseases); 5) reproductivedisorders (premature labor, dysmenorhea, preeclampsia) and 6)carcinogenesis. Rubanyi & Polokoff, supra.

Diseases can be evaluated for the impact of ET upon them byexamining: 1) increased production of ETs; 2) increased reactivity toETs; and/or 3) efficacy of an ET receptor antagonist, antibody or ECEinhibitor. Response to the previous criteria suggest that ETs likelyplay roles in cerebral vasospasm following subarachnoid hemorrhage,hypertension (fulminant complications), acute renal failure andcongestive heart failure. While inhibitors of ET production or activityhave not been used in models of coronary vasospasm, acute myocardialinfarction, and atherosclerosis, they do have elevated ET levels andincrease reactivity to ETs. Shock and pulmonary hypertension alsoexhibit elevated ET levels (Rubanyi and Polokoff, supra). Inhibition ofECEs in these conditions may be of therapeutic value.

The expression pattern of ECE-2 differs from that observed for ECE-1.ECE-2 was observed at low levels in the adult brain, lung and pancreasand higher levels in fetal brain and kidney by Northern blot analysis(FIG. 8). PCR revealed low levels of expression in additional tissues:fetal lung, fetal small intestine and adult testis. Fetal liver wasnegative. A similar pattern was reported for bovine ECE-2 (Emoto andYanangisawa, supra). It is expressed in brain tissues (cerebral cortex,cerebellum and adrenal medulla), myometrium and testis, and in lowlevels in ovary and very low levels in many other tissues. Bovine ECE-1(Xu et al, supra) is more widely and more abundantly expressed. It isobserved in vascular endothelial cells of most organs and in someparenchymal cells. With the exception for brain, bovine ECE-2 mRNA waspresent at lower levels than ECE-1. Applicants believe ECE-2 to be aparticularly good target for the therapeutic intervention for diseasessuch as cerebral vasospasm following subarachnoid hemorrhage and stroke.

Uses of the herein disclosed molecules may also be based upon thepositive functional assay hits disclosed and described below.

F. Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59–103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production ofhumanmonoclonalantibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51–631.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against PRO.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] s or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522–525 (1986); Riechmann etal., Nature, 332:323–329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593–596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522–525 (1986); Riechmann et al., Nature,332:323–327 (1988); Verhoeyen et al., Science, 239:1534–1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of ahumanantibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86–95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779–783(1992); Lonberg et al., Nature 368 856–859 (1994); Morrison, Nature 368,812–13 (1994); Fishwild et al., Nature Biotechnology 14, 845–51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65–93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537–539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J. 10:3655–3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217–225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547–1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444–6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).Antibodies with more tan two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO polypeptide herein. Alternatively, an anti-PRO polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRM (CD16) so as to focus cellular defense mechanisms to the cellexpressing the particular PRO polypeptide. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells which express aparticular PRO polypeptide. These antibodies possess a PRO-binding armand an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the PRO polypeptide and further binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191–1195(1992) and Shopes, J. Immunol. 148: 2918–2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560–2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219–230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phrytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniurn derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5difluoro-2,4 dinitrobenzene). For example, a ricin immunotoxin can beprepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286–288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19):1484 (1989).

9. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersin the form of pharmaceutical compositions.

If the PRO polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889–7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in Wvo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS-S bond formation through thiodisulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

G. Uses for Anti-PRO Antibodies

The anti-PRO antibodies of the invention have various utilities. Forexample, anti-PRO antibodies may be used in diagnostic assays for PRO,e.g., detecting its expression in specific cells, tissues, or serum.Various diagnostic assay techniques known in the art may be used, suchas competitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147–158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety Maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO antibodies also are useful for the affinity purification of PROfrom recombinant cell culture or natural sources. In this process, theantibodies against PRO are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the PROto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Rockville, Md.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 (Altschul and Gish, Methods in Enzymology 266: 460–480 (1996)) asa comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a Blast score of 70 (or insome cases 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences usingphrap. In addition, the consensus DNA sequences obtained were often (butnot always) extended using repeated cycles of BLAST and phrap to extendthe consensus sequence as far as possible using the sources of ESTsequences discussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward (.f) and reverse (.r) PCR primers generally rangefrom 20 to 30 nucleotides and are often designed to give a PCR productof about 100–1000 bp in length. The probe (.p) sequences are typically40–55 bp in length. In some cases, additional oligonucleotides aresynthesized when the consensus sequence is greater than about 1–1.5 kbp.In order to screen several libraries for a full-length clone, DNA fromthe libraries was screened by PCR amplification, as per Ausubel et al.,Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278–1280 (1991)) in theunique XhoI and NotI sites.

Example 2 Isolation of cDNA clones by Amylase Screening

1. Preparation of Olio dT Primed cDNA Library

mRNA was isolated from a human tissue of interest using reagents andprotocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNAwas used to generate an oligo dT primed cDNA library in the vector pRK5Dusing reagents and protocols from Life Technologies, Gaithersburg, Md.(Super Script Plasmid System). In this procedure, the double strandedcDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNAwas cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector thathas an sp6 transcription initiation site followed by an SfiI restrictionenzyme site preceding the XhoI/NotI cDNA cloning sites.

2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500–1000 bp, Tinkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g. CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD_(600=0.1)) into fresh YEPD broth (500ml) and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀ ₌0.4–0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol. <10 μl) in microfugetubes. The nixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mMLi₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5–10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208–210 (1994). Transformants were grown at 30° C. for 2–3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem., 172:176–179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50–100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (54μl) was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNfP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

-   5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′ (SEQ ID NO:324)

The sequence of reverse oligonucleotide 2 was:

-   5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′ (SEQ ID NO:325)

PCR was then performed as follows:

a. Denature 92° C.,  5 minutes b.  3 cycles of: Denature 92° C., 30seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c.  3 cyclesof: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C.,60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C.,30 seconds Extend 72° C., 60 seconds e. Hold  4° C.

The underlined regions of the oligonucleotides annealed to the ADHpromoter region and the amylase region, respectively, and amplified a307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3 Isolation of cDNA Clones Encoding Human PRO213

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA28735. Based on the DNA28735 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO213.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-TGGAGCAGCAATATGCCAGCC-3′ (SEQ ID NO:3)-   reverse PCR primer 5′-TTTTCCACTCCTGTCGGGTTGG-3′ (SEQ ID NO:4)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA28735 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GGTGACACTTGCCAGTCAGATGTGGATGAATGCAGTGCTAGGAGGG-3′ (SEQ ID NO:5)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO213 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO213 herein designated as UNQ187(DNA30943-1163)] (SEQ ID NO:1) and the derived protein sequence forPRO213.

The entire nucleotide sequence of UNQ187 (DNA30943-1163) is shown inFIG. 1 (SEQ ID NO:1). Clone UNQ187 (DNA30943-1163) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 336–338 and ending at the stop codon at nucleotidepositions 1221–1223 (FIG. 1). The predicted polypeptide precursor is 295amino acids long (FIG. 2). Clone UNQ187 (DNA30943-1163) has beendeposited with ATCC.

Analysis of the amino acid sequence of the full-length PRO213polypeptide suggests that a portion of it possesses significant homologyto the human growth arrest-specific gene 6 protein. More specifically,an analysis of the Dayhoff database (version 35.45 SwissProt 35)evidenced significant homology between the PRO213 amino acid sequenceand the following Dayhoff sequences, HSMHC3W5A_(—)6 and B48089.

Example 4 Isolation of cDNA Clones Encoding Human PRO274

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA36469. Based on the DNA36469 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO274. ESTsproprietary to Genentech were employed in the consensus assembly. TheESTs are shown in FIGS. 5–7 and are herein designated DNA17873, DNA36157and DNA28929, respectively.

Pairs of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 (36469.f1) 5′-CTGATCCGGTTCTTGGTGCCCCTG-3′ (SEQ    ID NO:11)-   forward PCR primer 2 (36469.f2) 5′-GCTCTGTCACTCACGCTC-3′ (SEQ ID    NO:12)-   forward PCR primer 3 (36469.f3) 5′-TCATCTCTTCCCTCTCCC-3′ (SEQ ID    NO:13)-   forward PCR primer 4 (36469.f4) 5′-CCTTCCGCCACGGAGTTC-3′ (SEQ ID    NO:14)-   reverse PCR primer 1 (36469.r1) 5′-GGCAAAGTCCACTCCGATGATGTC-3′ (SEQ    ID NO:15)-   reverse PCR primer 2 (36469.r2) 5′-GCCTGCTGTGGTCACAGGTCTCCG-3′ (SEQ    ID NO:16)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA36469 sequence which had the    following nucleotide sequence    hybridization probe (36469.p1)-   5′-TCGGGGAGCAGGCCTTGAACCGGGGCATTGCTGCTGTCAAGGAGG-3′ (SEQ ID NO:17)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO274 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO274 [herein designated as UNQ241(DNA39987-1184)] (SEQ ID NO:1) and the derived protein sequence forPRO274.

The entire nucleotide sequence of UNQ241 (DNA39987-1184) is shown inFIG. 3 (SEQ ID NO:6). Clone UNQ241 (DNA39987-1184) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 83–85 and ending at the stop codon at nucleotidepositions 1559–1561 (FIG. 3). The predicted polypeptide precursor is 492amino acids long (FIG. 4), has an estimated molecular weight of about54,241 daltons and an estimated pI of about 8.21. Clone UNQ241(DNA39987-1184) has been deposited with ATCC and is assigned ATCCdeposit no. 209786.

Analysis of the amino acid sequence of the full-length PRO274polypeptide suggests that it possesses significant homology to the Fn54protein. More specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35) evidenced significant homology between the PRO274amino acid sequence and the following Dayhoff sequences, MMFN54S2_(—)1,MMFN54S1_(—)1, CELF48C1_(—)8, CEF38B7_(—)6, PRP3_RAT, INL3_PIG,MTCY07A7_(—)13, YNAX_KLEAE, A47234 and HME2_MOUSE.

Example 5 Isolation of cDNA Clones Encoding Human PRO300

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA35930. Based on the DNA35930 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO300.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 1 (35930.f1) 5′-GCCGCCTCATCTTCACGTTCTTCC-3′ (SEQ    ID NO:20)-   forward PCR primer 2 (35930.f2) 5′-TCATCCAGCTGGTGCTGCTC-3′ (SEQ ID    NO:21)-   forward PCR primer 3 (35930.f3) 5′-CTTCTTCCACTTCTGCCTGG-3′ (SEQ ID    NO:22)-   forward PCR primer 4 (35930.f4) 5′-CCTGGGCAAAAATGCAAC-3′ (SEQ ID    NO:23)-   reverse PCR primer 1 (35930.r1) 5′-CAGGAATGTAGAAGGCACCCACGG-3′ (SEQ    ID NO:24)-   reverse PCR primer 2 (35930.r2) 5′-TGGCACAGATCTTCACCCACACGG-3′ (SEQ    ID NO:25)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA35930 sequence which had the    following nucleotide sequence    hybridization probe (35930.p1)-   5′-TGTCCATCATTATGCTGAGCCCGGGCGTGGAGAGTCAGCTCTACAAGCTG-3′ (SEQ ID    NO:26)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO300 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO300 [herein designated as UNQ263(DNA40625-1189)] (SEQ ID NO:18) and the derived protein sequence forPRO300.

The entire nucleotide sequence of UNQ263 (DNA40625-1189) is shown inFIG. 8 (SEQ ID NO:18). Clone UNQ263 (DNA40625-1189) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 45–47 and ending at the stop codon at nucleotidepositions 1416–1418 (FIG. 8). The predicted polypeptide precursor is 457amino acids long (FIG. 9). Clone UNQ263 (DNA40625-1189) has beendeposited with ATCC and is assigned ATCC deposit no. 209788.

Analysis of the amino acid sequence of the full-length PRO300polypeptide suggests that portions of it possess significant homology tothe Diff 33 protein. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO300 amino acid sequence and the following Dayhoffsequence, HSU49188_(—)1.

Example 6 Isolation of cDNA Clones Encoding Human PRO284

Two cDNA sequences were isolated in the amylase screen described inExample 2 and those cDNA sequences are herein designated DNA12982 (seeFIG. 12; human placenta-derived) and DNA15886 (see FIG. 13; humansalivary gland-derived). The DNA12982 and DNA15886 sequences were thenclustered and aligned, giving rise to a consensus nucleotide sequenceherein designated DNA18832.

Based on the DNA18832 consensus sequence, oligonucleotide probes weregenerated and used to screen a human placenta library (LIB89) preparedas described in paragraph 1 of Example 2 above. The cloning vector waspRK5B (pRK5B is a precursor of pRK5D that does not contain the SfiIsite; see, Holmes et al., Science, 253:1278–1280 (1991)), and the cDNAsize cut was less than 2800 bp.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 (18832.est.f) 5′-TCGTACAGTTACGCTCTCCC-3′ (SEQ    ID NO:31)-   forward PCR primer 2 (18832.f) 5′-CTTGAGGAGCGTCAGAAGCG-3′ (SEQ ID    NO:32)-   reverse PCR primer (18832.r) 5′-ATAACGAATGAAGCCTCGTG-3′ (SEQ ID    NO:33)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA18832 sequence which had the following    nucleotide sequence    hybridization probe (18832.p)-   5′-GCTAATATCTGTAAGACGGCAGCTACAGCAGGCATCATTG-3′ (SEQ ID NO:34)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO284 gene using the probe oligonucleotideand one of the PCR primers.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 167–169 and ending at the stop codon found at nucleotidepositions 1022–1024 (FIG. 10; SEQ ID NO:27). The predicted polypeptideprecursoris 285 amino acids long, has a calculated molecular weight ofapproximately 32,190 daltons and an estimated pI of approximately 9.03.Analysis of the full-length PRO284 sequence shown in FIG. 11 (SEQ IDNO:28) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 24, transmembrane domains fromabout amino acid 76 to about amino acid 96 and from about amino acid 171to about amino acid 195 and a potential N-glycosylation site from aboutamino acid 153 to about amino acid 156. Clone UNQ247 (DNA23318-1211) hasbeen deposited with ATCC on Apr. 21, 1998 and is assigned ATCC depositno. 209787.

Analysis of the amino acid sequence of the full-length PRO284polypeptide suggests that it possesses no significant sequencesimilarity to any known protein. However, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced some degree of homologybetween the PRO284 amino acid sequence and the following Dayhoffsequences, JQ0124, CELE04A4_(—)5, AB006451_(—)1, AF030162_(—)1,IM23_YEAST, S71194, NIA_CUCMA, IM17_YEAST, I50479 and HUMZFHP_(—)1.

Example 7 Isolation of cDNA Clones Encoding Human PRO296

A cDNA sequence isolated in the amylase screen as described in Example 2above was found, by BLAST and FastA sequence alignment, to have sequencehomology to a nucleotide sequence encoding sarcoma-associated proteinSAS. This cDNA sequence is herein designated DNA23020 (see FIG. 16). TheDNA23020 sequence was then compared to a variety of expressed sequencetag (EST) databases which included public EST databases (e.g., GenBank)and a proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals,Palo Alto, Calif.) to identify existing homologies. The homology searchwas performed using the computer program BLAST or BLAST2 (Altshul etal., Methods in Enzymology 266:460–480 (1996)). Those comparisonsresulting in a BLAST score of 70 (or in some cases 90) or greater thatdid not encode known proteins were clustered and assembled into aconsensus DNA sequence with the program “phrap” (Phil Green, Universityof Washington, Seattle, Wash.). The consensus sequence obtainedtherefrom is herein designated DNA35858. Two proprietary Genentech ESTswere employed in the assembly wherein those EST sequences are hereinidentified as DNA21971 (FIG. 17; SEQ ID NO:38) and DNA29037 (FIG. 18;SEQ ID NO:39).

Based on the DNA35858 consensus sequence, oligonucleotide probes weregenerated and used to screen a human kidney library (LIB3228) libraryprepared as described in paragraph 1 of Example 2 above. The cloningvector was pRK5B (pRK5B is a precursor of pRK5D that does not containthe SfiI site; see, Holmes et al., Science, 253:1278–1280 (1991)), andthe cDNA size cut was less than 2800 bp.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 (35858.f1) 5′-ACCCACGTCTGCGTTGCTGCC-3′ (SEQ ID    NO:40)-   forward PCR primer 2 (35858.f2) 5′-GAGAATATGCTGGAGAGG-3′ (SEQ ID    NO:41)-   reverse PCR primer (35858.r1) 5′-AGGAATGCACTAGGATTCGCGCGG-3′ (SEQ ID    NO:42)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA35858 sequence which had the    following nucleotide sequence    hybridization probe (35858.p1)-   5′-GGCCCCAAAGGCAAGGACAAAGCAGCTGTCAGGGAACCTCCGCCG-3′ (SEQ ID NO:43)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO296 gene using the probe oligonucleotideand one of the PCR primers.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 174–176 and ending at the stop codon found at nucleotidepositions 786–788 (FIG. 14; SEQ ID NO:35). The predicted polypeptideprecursor is 204 amino acids long, has a calculated molecular weight ofapproximately 22,147 daltons and an estimated pI of approximately 8.37.Analysis of the full-length PRO296 sequence shown in FIG. 15 (SEQ IDNO:36) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 34 and transmembrane domains fromabout amino acid 47 to about amino acid 63, from about amino acid 72 toabout amino acid 95 and from about amino acid 162 to about amino acid182. Clone UNQ260 (DNA39979-1213) has been deposited with ATCC on Apr.21, 1998 and is assigned ATCC deposit no. 209789.

Analysis of the amino acid sequence of the full-length PRO296polypeptide suggests that it possesses significant sequence similarityto the sarcoma-amplified SAS protein, thereby indicating that PRO296 maybe a novel SAS homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO296 amino acid sequence and the following Dayhoffsequences, I58391, GEN11061, SSC2B04_(—)1, HSU81031_(—)2, CD63_RAT,CD63_MOUSE, CD63_HUMAN, AF022813_(—)1, CD63_RABIT and CO02_HUMAN.

Example 8 Isolation of cDNA Clones Encoding Human PRO329

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA35612. Based on the DNA35612 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO329.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 (35612.f1) 5′-TGGGCTGTGTCCTCATGG-3′ (SEQ ID    NO:46)-   forward PCR primer 2 (35612.f2) 5′-TTTCCAGCGCCAATTCTC-3′ (SEQ ID    NO:47)-   reverse PCR primer 1 (35612.r1) 5′-AGTTCTTGGACTGTGATAGCCAC-3′ (SEQ    ID NO:48)-   reverse PCR primer 2 (35612.r2) 5′-AAACTTGGTTGTCCTCAGTGGCTG-3′ (SEQ    ID NO:49)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA35612 sequence which had the    following nucleotide sequence    hybridization probe (35612.p1)-   5′-GTGAGGGACCTGTCTGCACTGAGGAGAGCAGCTGCCACACGGAGG-3′ (SEQ ID NO:50)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO329 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB6).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO329 [herein designated as UNQ291(DNA40594-1233)] (SEQ ID NO:44) and the derived protein sequenceforPRO329.

The entire nucleotide sequence of UNQ291 (DNA40594-1233) is shown inFIG. 19 (SEQ ID NO:44). Clone UNQ291 (DNA40594-1233) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 9–11 and ending at the stop codon at nucleotidepositions 1086–1088 (FIG. 19). The predicted polypeptide precursor is359 amino acids long (FIG. 20). The full-length PRO329 protein shown inFIG. 20 has an estimated molecular weight of about 38,899 daltons and apI of about 5.21. Clone UNQ291 (DNA40594-1233) has been deposited withATCC on Feb. 5, 1998 and is assigned ATCC deposit no. 209617.

Analysis of the amino acid sequence of the full-length PRO329polypeptide suggests that it possesses significant sequence similarityto a high affinity immunoglobulin F_(c) receptor protein. Morespecifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant homology between the PRO329 aminoacid sequence and the following Dayhoff sequences, FCG1_HUMAN,FCG0_HUMAN, P_R91439, P_R22549, P_R91438, P_W00859, P_R20811, P_R22550,HUMCD6406_(—)1 and FCG1_MOUSE.

Example 9 Isolation of cDNA Clones Encoding Human PRO362

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42257. Based on the DNA42257 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO362.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 (42257.f1) 5′-TATCCCTCCAATTGAGCACCCTGG-3′ (SEQ    ID NO:53)-   forward PCR primer 2 (42257.f2) 5′-GTCGGAAGACATCCCAACAAG-3′ (SEQ ID    NO:54)-   reverse PCR primer 1 (42257.r1) 5′-CTTCACAATGTCGCTGTGCTGCTC-3′ (SEQ    ID NO:55)-   reverse PCR primer 2 (42257.r2) 5′-AGCCAAATCCAGCAGCTGGCTTAC-3′ (SEQ    ID NO:56)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA42257 sequence which had the    following nucleotide sequence    hybridization probe (42257.p1)-   5′-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCCTGAT-3′ (SEQ ID    NO:57)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO362 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal brain tissue (LIB153).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO362 [herein designated as UNQ317(DNA45416-1251)] (SEQ ID NO:51) and the derived protein sequence forPRO362.

The entire nucleotide sequence of UNQ317 (DNA45416-1251) is shown inFIG. 21 (SEQ ID NO:51). Clone UNQ317 (DNA45416-1251) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 119–121 and ending at the stop codon at nucleotidepositions 1082–1084 (FIG. 21). The predicted polypeptide precursor is321 amino acids long (FIG. 22). The full-length PRO362 protein shown inFIG. 2 has an estimated molecular weight of about 35,544 daltons and apI of about 8.51. Analysis of the full-length PRO362 polypeptide asshown in FIG. 22 evidences the presence of a glycosaminoglycanattachment site at about amino acid 149 to about amino acid 152 and atransmembrane domain from about amino acid 276 to about amino acid 306.Clone UNQ317 (DNA45416-1251) has been deposited with ATCC on Feb. 5,1998 and is assigned ATCC deposit no. 209620.

Analysis of the amino acid sequence of the full-length PRO362polypeptide suggests that it possesses significant sequence similarityto the A33 antigen protein and the HCAR protein. More specifically, ananalysis of the Dayhoff database (version 35.45 SwissProt 35) evidencedsignificant homology between the PRO362 amino acid sequence and thefollowing Dayhoff sequences, AB002341_(—)1, HSU55258_(—)1,HSC7NRCAM_(—)1, RNU81037_(—)1, A33_HUMAN, P_W14158, NMNCAMRI_(—)1,HSTITINN2_(—)1, S71824_(—)1 and HSU63041_(—)1.

Example 10 Isolation of cDNA Clones Encoding Human PRO363

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42828. Based on the DNA42828 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO363.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer (42828.f1) 5′-CCAGTGCACAGCAGGCAACGAAGC-3′ (SEQ ID    NO:60)-   reverse PCR primer (42828.r1) 5′-ACTAGGCTGTATGCCTGGGTGGGC-3′ (SEQ ID    NO:61)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA42828 sequence which had the    following nucleotide sequence    hybridization probe (42828.p1)-   5′-GTATGTACAAAGCATCGGCATGGTTGCAGGAGCAGTGACAGGC-3′ (SEQ ID NO:62)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO363 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO363 [herein designated as UNQ318(DNA45419-1252)] (SEQ ID NO:58) and the derived protein sequence forPRO363.

The entire nucleotide sequence of UNQ318 (DNA45419-1252) is shown inFIG. 23 (SEQ ID NO:58). Clone UNQ318 (DNA45419-1252) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 190–192 and ending at the stop codon at nucleotidepositions 1309–1311 (FIG. 23). The predicted polypeptide precursor is373 amino acids long (FIG. 24). The full-length PRO363 protein shown inFIG. 24 has an estimated molecular weight of about 41,281 daltons and apI of about 8.33. A transmembrane domain exists at amino acids 221 to254 of the amino acid sequence shown in FIG. 24 (SEQ ID NO:59). ThePRO363 polypeptide also possesses at least two myelin P0 protein domainsfrom about amino acids 15 to 56 and from about amino acids 87 to 116.Clone UNQ318 (DNA45419-1252) has been deposited with ATCC on Feb. 5,1998 and is assigned ATCC deposit no. 209616.

Analysis of the amino acid sequence of the full-length PRO363polypeptide suggests that it possesses significant sequence similarityto the cell surface protein HCAR, thereby indicating that PRO363 may bea novel HCAR homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO363 amino acid sequence and the following Dayhoffsequences, HS46KDA_(—)1, HSU90716_(—)1, MMCARH_(—)1, MMCARHOM_(—)1,MMU90715_(—)1, A33_HUMAN, P_W14146, P_W14158, A42632 and B42632.

Example 11 Isolation of cDNA Clones Encoding Human PRO868

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA38133. Based on the DNA38133 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO868.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer (38133.f1) 5′-GTAGCAGTGCACATGGGGTGTTGG-3′ (SEQ ID    NO:65)-   reverse PCR primer (38133.r1) 5′-ACCGCACATCCTCAGTCTCTGTCC-3′ (SEQ ID    NO:66)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA38133 sequence which had the    following nucleotide sequence    hybridization probe (38133.p1)-   5′-ACGATGATCGCGGGCTCCCTTCTCCTGCTTGGATTCCTTAGCACCACCAC-3′ (SEQ ID    NO:67)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO868 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO868 [herein designated as UNQ437(DNA52594-1270)] (SEQ ID NO:63) and the derived protein sequence forPRO868.

The entire nucleotide sequence of UNQ437 (DNA52594-1270) is shown inFIG. 25 (SEQ ID NO:63). Clone UNQ437 (DNA52594-1270) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 325–327 and ending at the stop codon at nucleotidepositions 2290–2292 FIG. 25). The predicted polypeptide precursor is 655amino acids long (FIG. 26). The full-length PRO868 protein shown in FIG.26 has an estimated molecular weight of about 71,845 daltons and a pI ofabout 8.22. Analysis of the full-length PRO868 polypeptide sequencedemonstrates the presence of conserved cysteine-containing domains fromabout amino acid 66 to about amino acid 78 and from about amino acid 123to about amino acid 134 of the sequence shown in FIG. 26 (SEQ ID NO:3),a TNFR death domain from about amino acid 85 to about amino acid 110, aFASA_mouse death domain block from about amino acid 159 to about aminoacid 175 and a transmembrane domain from about amino acid 347 to aboutamino acid 375. Clone UNQ437 (DNA52594-1270) has been deposited withATCC on Mar. 17, 1998 and is assigned ATCC deposit no. 209679

Analysis of the amino acid sequence of the full-length PRO868polypeptide suggests that it possesses significant sequence similarityto the tumor necrosis factor receptor protein, thereby indicating thatPRO868 may be a novel member of the tumor necrosis factor receptorfamily. More specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35) evidenced significant homology between the PRO868amino acid sequence and the following Dayhoff sequences, RNU94330_(—)1,P_R99933, PR_R99945, P_R99950, HSU94332_(—)1, CD40_HUMAN, S63368_(—)1,TNR2_HUMAN, MVU87844_(—)1 AND CVU87837_(—)1.

Example 12 Isolation of cDNA Clones Encoding Human PRO382

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA30892. Based on the DNA30892 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO382.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-TGACATCGCCCTTATGAAGCTGGC-3′ (SEQ ID NO:70)-   reverse PCR primer 5′-TACACGTCCCTGTGGTTGCAGATC-3′ (SEQ ID NO:71)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA30892 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CGTTCAATGCAGAAATGATCCAGCCTGTGTGCCTGCCCAACTCTGAAGAG-3′ (SEQ ID    NO:72)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO382 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO382 [herein designated as UNQ323(DNA45234-1277)] (SEQ ID NO:68) and the derived protein sequence forPRO382.

The entire nucleotide sequence of UNQ323 (DNA45234-1277) is shown inFIG. 27 (SEQ ID NO:68). Clone UNQ323 (DNA45234-1277) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 126–128 and ending at the stop codon at nucleotidepositions 1485–1487 (FIG. 27). The predicted polypeptide precursor is453 amino acids long (FIG. 28). The full-length PRO382 protein shown inFIG. 28 has an estimated molecular weight of about 49,334 daltons and apI of about 6.32. Analysis of the native PRO382 amino acid sequenceshown in FIG. 28 (SEQ ID NO:69) indicates the presence of a putativetransmembrane domain from about amino acid 240 to about amino acid 284,a putative signal peptide at about amino acid 1 to about amino acid 20,a putative apple domain at about amino acid 386 to about amino acid 419,a putative Kringle domain at about amino acid 394 to about amino acid406 and a histidine-containing protease active site at about amino acid253 to about amino acid 258. Clone UNQ323 (DNA45234-1277) has beendeposited with ATCC on Mar. 5, 1998 and is assigned ATCC deposit no.209654.

Analysis of the amino acid sequence of the full-length PRO382polypeptide suggests that it possess significant homology to serineprotease proteins, thereby indicating that PRO382 may be a novel serineprotease. Specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35) evidenced significant homology between the PRO382amino acid sequence and the following Dayhoff sequences, HSU75329_(—)1,ENTK_MOUSE, HEPS_HUMAN, AF030065_(—)1, HEPS_RAT, PLMN_PIG, P_R89430,P_R89435, PLMN_HORSE, PLMN_BOVIN and P_R83959.

Example 13 Isolation of cDNA Clones Encoding Human PRO545

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA44706. An EST proprietary to Genentech wasemployed in the consensus assembly and is herein designated DNA13217(FIG. 31; SEQ ID NO:75). Based on the DNA44706 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO545.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 1 5′-GTCTCAGCACGTGTTCTGGTCTCAGGG-3′ (SEQ ID    NO:76)-   forward PCR primer 2 5′-CATGAGCATGTGCACGGC-3′ (SEQ ID NO:77)-   forward PCR primer 3 5′-TACCTGCACGATGGGCAC-3′ (SEQ ID NO:78)-   forward PCR primer 4 5′-CACTGGGCACCTCCCTTC-3′ (SEQ ID NO:79)-   reverse PCR primer 1 5′-CTCCAGGCTGGTCTCCAAGTCCTTCC-3′ (SEQ ID NO:80)-   reverse PCR primer 2 5′-TCCCTGTTGGACTCTGCAGCTTCC-3′ (SEQ ID NO:81)-   reverse PCR primer 3 5′-CTTCGCTGGGAAGAGTTTG-3′ (SEQ ID NO:82)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA44706 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GTGCAACCAACAGATACAAACTCTTCCCAGCGAAGAAGCTGAAAAGCGTC-3′ (SEQ ID    NO:83)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO545 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human placenta tissue (LIB90).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO545 [herein designated as UNQ346(DNA49624-1279)] (SEQ ID NO:73) and the derived protein sequence forPRO545.

The entire nucleotide sequence of UNQ346 (DNA49624-1279) is shown inFIG. 29 (SEQ ID NO:73). Clone UNQ346 (DNA49624-1279) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 311–313 and ending at the stop codon at nucleotidepositions 2516–2518 (FIG. 29). The predicted polypeptide precursor is735 amino acids long (FIG. 30). The full-length PRO545 protein shown inFIG. 30 has an estimated molecular weight of about 80,177 daltons and apI of about 7.08. Important regions of the PRO545 amino acid sequenceinclude the signal peptide, corresponding to amino acids 1–28, fivepotential N-glycosylation sites, from about amino acid 111–114, aminoacids 146–149, amino acids 348–351, amino acids 449–452, and amino acids648–651, and a neutral zinc metallopeptidase, zinc-binding regionsignature sequence, from about amino acids 344–353. Clone UNQ346(DNA49624-1279) has been deposited with ATCC and is assigned ATCCdeposit no. 209655.

Example 14 Isolation of cDNA Clones Encoding Human PRO617

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42798. Based on the DNA42798 sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO617.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-ACGGGCACACTGGATCCCAAATG-3′ (SEQ ID NO:86)-   reverse PCR primer 5′-GGTAGAGATGTAGAAGGGCAAGCAAGACC-3′ (SEQ ID    NO:87)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA42798 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GCTCCCTACCCGTGCAGGTTTCTTCATTTGTTCCTTTAACCAGTATGCCG-3′ (SEQ ID    NO:88)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO617 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO617 [herein designated as UNQ353(DNA48309-1280)] (SEQ ID NO:1) and the derived protein sequence forPRO617.

The entire nucleotide sequence of UNQ353 (DNA48309-1280) is shown inFIG. 32 (SEQ ID NO:84). Clone UNQ353 (DNA48309-1280) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 723–725 and ending at the stop codon at nucleotidepositions 924–926 (FIG. 32). The predicted polypeptide precursor is 67amino acids long (FIG. 33). The full-length PRO617 protein shown in FIG.33 has an estimated molecular weight of about 6,981 daltons and a pI ofabout 7.47. Analysis of the PRO617 amino acid sequence also evidencesthe existence of a putative signal peptide from about amino acid 15 toabout amino acid 27 and a putative protein kinase C phosphorylation sitefrom about amino acid 41 to about amino acid 43. Clone UNQ353(DNA48309-1280) has been deposited on Mar. 5, 1998 with ATCC and isassigned ATCC deposit no. 209656.

Analysis of the amino acid sequence of the full-length PRO617polypeptide suggests that it possesses significant homology to the CD24protein, thereby indicating that PRO617 may be a novel CD24 homolog.More specifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant homology between the PRO617 aminoacid sequence and the following Dayhoff sequences, CD24_HUMAN,CD24_MOUSE, S15785, CD24_RAT, VGE BPG4, MSE5_HUMAN, HSMHC3W36A_(—)2,MLU15184_(—)8, P R85075, SEPL_HUMAN and MTCY63_(—)13.

Example 15 Isolation of cDNA Clones Encoding Human PRO700

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA30837. Based on the DNA30837 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO700.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 1 5′-ATGTTCTTCGCGCCCTGGTG-3′ (SEQ ID NO:91)-   forward PCR primer 2 5′-CCAAGCCAACACACTCTACAG-3′ (SEQ ID NO:92)-   reverse PCR primer 1 5′-AAGTGGTCGCCTTGTGCAACGTGC-3′ (SEQ ID NO:93)-   reverse PCR primer 2 5′-GGTCAAAGGGGATATATCGCCAC-3′ (SEQ ID NO:94)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA30837 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GCATGGAAGATGCCAAAGTCTATGTGGCTAAAGTGGACTGCACGGCCCA-3′ (SEQ ID    NO:95)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO700 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO700 [herein designated as UNQ364(DNA46776-1284)] (SEQ ID NO:89) and the derived protein sequence forPRO700.

The entire nucleotide sequence of UNQ364 (DNA46776-1284) is shown inFIG. 34 (SEQ ID NO:89). Clone UNQ364 (DNA46776-1284) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 33–35 and ending at the stop codon at nucleotidepositions 1329–1331 (FIG. 34). The predicted polypeptide precursor is432 amino acids long (FIG. 35). The full-length PRO700 protein shown inFIG. 35 has an estimated molecular weight of about 47,629 daltons and apI of about 5.90. Important regions of the amino acid sequence of PRO700include the signal peptide, corresponding to amino acids from about 1 to33, regions homologous to disulfide isomerase, corresponding to aminoacids from about 82–99, 210–255, and 345–360, a tyrosine kinasephosphorylation site, corresponding to amino acids from about 143–151,and an endoplasmic reticulum targeting sequence, corresponding to aminoacids from about 429–432. Clone UNQ364 (DNA46776-1284) has beendeposited with ATCC and is assigned ATCC Deposit No. 209721.

Example 16 Isolation of cDNA Clones Encoding Human PRO702

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA36623. Based on the DNA36623 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO702.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer (36623.f1) 5′-CGCTGACTATGTTGCCAAGAGTGG-3′ (SEQ ID    NO:98)-   reverse PCR primer (36623.r1) 5′-GATGATGGAGGCTCCATACCTCAG-3′ (SEQ ID    NO:99)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA36623 sequence which had the    following nucleotide sequence    hybridization probe (36623.p1)-   5′-GTGTTCATTGGCGTGAATGACCTTGAAAGGGAGGGACAGTACATGTTCAC-3′ (SEQ ID    NO:100)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO702 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO702 [herein designated as UNQ366(DNA50980-1286)] (SEQ ID NO:96) and the derived protein sequence forPRO702.

The entire nucleotide sequence of UNQ366 (DNA50980-1286) is shown inFIG. 36 (SEQ ID NO:96). Clone UNQ366 (DNA50980-1286) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 22–24 and ending at the stop codon at nucleotidepositions 853–855 (FIG. 36). The predicted polypeptide precursor is 277amino acids long (FIG. 37). The full-length PRO702 protein shown in FIG.37 has an estimated molecular weight of about 30,645 daltons and a pI ofabout 7.47. Analysis of the full-length native PRO702 amino acidsequence evidences the presence of a putative signal peptide from aboutamino acid 1 to about amino acid 25, potential N-glycosylation sitesfrom about amino acid 230 to about amino acid 233 and from about aminoacid 258 to about amino acid 261 and a C-type lectin domain signaturesequence from about amino acid 248 to about amino acid 270. Clone UNQ366(DNA50980-1286) has been deposited with ATCC on Mar. 31, 1998 and isassigned ATCC deposit no. 209717.

Analysis of the amino acid sequence of the full-length PRO702polypeptide suggests that it possesses significant sequence similarityto the conglutinin protein, thereby indicating that PRO702 may be anovel conglutinin homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO702 amino acid sequence and the following Dayhoffsequences, S32436, P_R75642, P_W18780, P_W18781, A53330, AC002528_(—)1,HSPPA2IC0_(—)1, CA21_HUMAN, CA14_HUMAN and A61262.

Example 17 Isolation of cDNA Clones Encoding Human PRO703

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43047. Based on the DNA43047 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO703.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-GAGAGCCATGGGGCTCCACCTG-3′ (SEQ ID NO:103)-   reverse PCR primer 1 5′-GGAGAATGTGGCCACAAC-3′ (SEQ ID NO:104)-   reverse PCR primer 2 5′-GCCCTGGCACAGTGACTCCATAGACG-3′ (SEQ ID    NO:105)-   reverse PCR primer 3 5′-ATCCACTTCAGCGGACAC-3′ (SEQ ID NO:106)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA40654 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CCAGTGCCAGGATACCTCTCTTCCCCCCAGAGCATAACAGACACG-3′ (SEQ ID NO:107)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO703 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO703 [herein designated as UNQ367(DNA50913-1287)] (SEQ ID NO:101) and the derived protein sequence forPRO703.

The entire nucleotide sequence of UNQ367 (DNA50913-1287) is shown inFIG. 38 (SEQ ID NO:101). Clone UNQ367 (DNA50913-1287) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 115–117 and ending at the stop codon at nucleotidepositions 2305–2307 (FIG. 38). The predicted polypeptide precursor is730 amino acids long (FIG. 39). The full-length PRO703 protein shown inFIG. 39 has an estimated molecular weight of about 78,644 daltons, and apI of about: 7.65. Important regions of the PRO703 amino acid sequenceinclude the signal peptide, a cAMP- and cGMP-dependent protein kinasephosphorylation site, a CUB domain protein motif, N-glycosylation sitesand a putative AMP-binding domain signature. Clone UNQ367(DNA50913-1287) has been deposited with ATCC and is assigned ATCCdeposit no. 209716.

Example 18 Isolation of cDNA Clones Encoding Human PRO705

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43437. Based on the DNA43437 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO705.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-AAGCGTGACAGCGGGCACGTC-3′ (SEQ ID NO:110)-   reverse PCR primer 5′-TGCACAGTCTCTGCAGTGCCCAGG-3′ (SEQ ID NO:111)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA43437 sequence which had the    following nucleotide sequence    hybridization probe (43437.p1)-   5′-GAATGCTGGAACGGGCACAGCAAAGCCAGATACTTGCCTG-3′ (SEQ ID NO:112)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO705 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO705 [herein designated as UNQ369(DNA50914-1289)] (SEQ ID NO:108) and the derived protein sequence forPRO705.

The entire nucleotide sequence of UNQ369 (DNA50914-1289) is shown inFIG. 40 (SEQ ID NO:108). Clone UNQ369 (DNA50914-1289) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 566–568 and ending at the stop codon at nucleotidepositions 2231–2233 (FIG. 40). The predicted polypeptide precursor is555 amino acids long (FIG. 41). The full-length PRO705 protein shown inFIG. 41 has an estimated molecular weight of about 62,736 daltons and apI of about 5.36. Analysis of the full-length PRO705 sequence as shownin FIG. 41 evidences the presence of the following: a signal peptidefrom about amino acid 1 to about amino acid 23, a eukaryotic DNAtopoisomerase 1 active site from about amino acid 418 to about aminoacid 436, and various regions that show homology to various glypicanproteins from about amino acid 237 to about amino acid 279, about aminoacid 421 to about amino acid 458, about amino acid 53 to about aminoacid 74, about amino acid 466 to about amino acid 504, about amino acid308 to about amino acid 355, about amino acid 104 to about amino acid156 and about amino acid 379 to about amino acid 410. Clone UNQ369(DNA50914-1289) has been deposited with ATCC on Mar. 31, 1998 and isassigned ATCC deposit no. 209722.

Analysis of the amino acid sequence of the full-length PRO705polypeptide suggests that it possesses significant sequence similarityto the K-glypican protein, thereby indicating that PRO705 may be a novelglypican protein family member. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO705 amino acid sequence and the followingDayhoff sequences, GPCK_MOUSE, GLYP_CHICK, GLYP_RAT, GLYP_HUMAN,GPC2_RAT, GPC5_HUMAN, GPC3_HUMAN, GPC3_RAT, P_(—R)30168 andCEC03H12_(—)2.

Example 19 Isolation of cDNA Clones Encoding Human PRO708

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA34024. Based on the DNA34024 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO708.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CCCAACCCAACTGTTTACCTCTGG-3′ (SEQ ID NO:115)

reverse PCR primer 5′-CTCTCTGAGTGTACATCTGTGTGG-3′ (SEQ ID NO:116)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA34024 sequence which had the followingnucleotide sequence

hybridization probe

-   5′-GCCACCCTACCTCAGAAACTGAAGGAGG NTATTCAACGCATATGGTCGG-3′ (SEQ ID    NO:117)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO708 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human bone marrow tissue (LIB255).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO708 herein designated as UNQ372(DNA48296-1292)] (SEQ ID NO:113) and the derived protein sequence forPRO708.

The entire nucleotide sequence of UNQ372 (DNA48296-1292) is shown inFIGS. 42A–B (SEQ ID NO:113). Clone UNQ372 (DNA48296-1292) contains asingle open reading frame with an apparent translational initiation siteat nucleotide positions 891–893 and ending at the stop codon atnucleotide positions 2436–2438 (FIGS. 42A–B). The predicted polypeptideprecursor is 515 amino acids long (FIG. 43). The full-length PRO708protein shown in FIG. 43 has an estimated molecular weight of about56,885 daltons and a pI of about 6.49. Analysis of the PRO708 amino acidsequence shown in FIG. 43 (SEQ ID NO:114) evidences the existence of aputative signal peptide at about amino acid 1 to about amino acid 37,putative sulfatase signature sequences at about amino acid 120 to aboutamino acid 132 and about amino acid 168 to about amino acid 177, aputative tyrosine kinase phosphorylation site from about amino acid 163to about amino acid 169 and potential N-glycosylation sites from aboutamino acid 157 to about amino acid 160, about amino acid 306 to aboutamino acid 309 and about amino acid 318 to about amino acid 321. CloneUNQ372 (DNA48296-1292) has been deposited with ATCC on Mar. 11, 1998 andis assigned ATCC deposit no. 209668.

Analysis of the amino acid sequence of the full-length PRO708polypeptide suggests that it possesses significant homology to the arylsulfatase proteins, thereby indicating that PRO708 may be a novel arylsulfatase homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO708 amino acid sequence and the following Dayhoffsequences, ARSB_HUMAN, CELC54D2_(—)2, G02857, STS_HUMAN, I37186, I37187,GEN12648, CELD1014_(—)7, GA6S_HUMAN and SPHM_HUMAN.

Example 20 Isolation of cDNA Clones Encoding Human PRO320

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA28739. Based on the DNA28739 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO320.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CCTCAGTGGCCACATGCTCATG-3′ (SEQ ID NO:120)-   reverse PCR primer 5′-GGCTGCACGTATGGCTATCCATAG-3′ (SEQ ID NO:121)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA28739 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GATAAACTGTCAGTACAGCTGTGAAGACACAGAAGAAGGGCCACAGTGCC-3′ (SEQ ID    NO:122)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO320 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue (LIB25).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO320 [herein designated as UNQ281(DNA32284-1307)] (SEQ ID NO:118) and the derived protein sequence forPRO320.

The entire nucleotide sequence of UNQ281 (DNA32284-1307) is shown inFIG. 44 (SEQ ID NO:118). Clone UNQ281 (DNA32284-1307) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 135–137 and ending at the stop codon at nucleotidepositions 1149–1151 (FIG. 44). The predicted polypeptide precursor is338 amino acids long (FIG. 45). The full-length PRO320 protein shown inFIG. 45 has an estimated molecular weight of about 37,143 daltons and apI of about 8.92. Important regions of the PRO320 amino acid sequenceinclude the signal peptide, corresponding to amino acids 1–21, anEGF-like domain cysteine pattern signature, corresponding to amino acids80–91, and three calcium-binding EGF-like domains, corresponding toamino acids 103–124, 230–151 and 185–206, respectively. Clone UNQ281(DNA32284-1307) has been deposited with ATCC and is assigned ATCCdeposit no. 209670.

Example 21 Isolation of cDNA Clones Encoding Human PRO324

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA34347. Based on the DNA34347 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO324.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 5′-GCAATGAACTGGGAGCTGC-3′ (SEQ ID NO:125)-   forward PCR primer 2 5′-CTGTGAATAGCATCCTGGG-3′ (SEQ ID NO:126)-   forward PCR primer 3 5′-CTTTTCAAGCCACTGGAGGG-3′ (SEQ ID NO:127)-   reverse PCR primer 1 5′-CTGTAGACATCCAAGCTGGTATCC-3′ (SEQ ID NO:128)-   reverse PCR primer 2 5′-AAGAGTCTGCATCCACACCACTC-3′ (SEQ ID NO:129)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA34347 sequence which had the    following nucleotide sequence    hybridization probe-   5′-ACCTGACGCTACTATGGGCCGAGTGGCAGGGACGACGCCCAGAATG-3′ (SEQ ID NO:130)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO324 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal liver tissue (LIB6).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO324 [herein designated as UNQ285(DNA36343-1310)] (SEQ ID NO:123) and the derived protein sequence forPRO324.

The entire nucleotide sequence of UNQ285 (DNA36343-1310) is shown inFIG. 46 (SEQ ID NO:123). Clone UNQ285 (DNA36343-1310) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 144–146 and ending at the stop codon at nucleotidepositions 1011–1013 (FIG. 46). The predicted polypeptide precursor is289 amino acids long (FIG. 47). The full-length PRO324 protein shown inFIG. 47 has an estimated molecular weight of about 32,268 daltons and apI of about 9.21. Analysis of the PRO324 polypeptide sequence shown inFIG. 47 (SEQ ID NO:124) evidence the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 31, a transmembranedomain from about amino acid 136 to about amino acid 157, tyrosinekinase phosphorylation sites from about amino acid 106 or about aminoacid 107 to about amino acid 113 and regions that are homologous toshort-chain alcohol dehydrogenase regions from about amino acid 80 toabout amino acid 90, from about amino acid 131 to about amino acid 168,from about amino acid 1 to about amino acid 13 and from about amino acid176 to about amino acid 185. Clone UNQ285 (DNA36343-1310) has beendeposited with ATCC on Mar. 30, 1998 and is assigned ATCC deposit no.209718.

Analysis of the amino acid sequence of the full-length PRO324polypeptide suggests that it possesses significant sequence similarityto oxidoreductases, thereby indicating that PRO324 may be a noveloxidoreductase homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO324 amino acid sequence and the following Dayhoffsequences, B61209, A69965, YQJQ_BACSU, D69930, S76124, FABG_(—) ECOLI,C70023, S77280, FABG_VIBHA and MTV013_(—)6.

Example 22 Isolation of cDNA Clones Encoding Human PRO351

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA35950. Based on the DNA35950 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO351.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-CCTGTGCTGTGCCTCGAGCCTGAC-3′ (SEQ ID NO:133)-   reverse PCR primer 5′-GTGGGCAGCAGTTAGCACCGCCTC-3′ (SEQ ID NO:134)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA35950 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GGCTGGCATCATCAGCTTTGCATCAAGCTGTGCCCAGGAGGACGC-3′ (SEQ ID NO:135)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO351 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal liver tissue (LIB230).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO351 [herein designated as UNQ308(DNA40571-1315)] (SEQ ID NO:131) and the derived protein sequence forPRO351.

The entire nucleotide sequence of UNQ308 (DNA40571-1315) is shown inFIG. 48 (SEQ ID NO:131). Clone UNQ308 (DNA40571-1315) contains two openreading frames with an apparent translational initiation site atnucleotide positions 189–191 and a second open reading frame beginningat nucleotide 470, with the two open reading frames ending at the stopcodons at nucleotide positions 363–365 and 2009–2011, respectively (FIG.48). The predicted polypeptide precursor is 571 amino acids long (FIG.49). Important regions of the amino acid sequence of PRO351 include thesignal peptide, regions having sequence similarity to serine proteasesof the trypsin family, two N-glycosylation sites, and three Kringledomains. Clone UNQ308 (DNA40571-1315) has been deposited with ATCC andis assigned ATCC deposit no. 209784.

Example 23 Isolation of cDNA Clones Encoding Human PRO352

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA36950. Based on the DNA36950 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO352.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 5′-CTGGCACAGCTCAACCTCATCTGG-3′ (SEQ ID NO:138)-   forward PCR primer 2 5′-GCTGTCTGTCTGTCTCATTG-3′ (SEQ ID NO:139)-   forward PCR primer 3 5′-GGACACAGTATACTGACCAC-3′ (SEQ ID NO:140)-   reverse PCR primer 1 5′-TGCGAACCAGGCAGCTGTAAGTGC-3′ (SEQ ID NO:141)-   reverse PCR primer 2 5′-TGGAAGAAGAGGGTGGTGATGTGG-3′ (SEQ ID NO:142)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA36950 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CAGCTGACAGACACCAAACAGCTGGTGCACAGTTTCACCGAAGGC-3′ (SEQ ID NO:143)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO352 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO352 [herein designated as UNQ309(DNA41386-1316)1 (SEQ ID NO:136) and the derived protein sequence forPRO352.

The entire nucleotide sequence of UNQ309 (DNA41386-1316) is shown inFIG. 50 (SEQ ID NO:136). Clone UNQ309 (DNA41386-1316) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 152–154 and ending at the stop codon at nucleotidepositions 1100–1102 (FIG. 50). The predicted polypeptide precursor is316 amino acids long (FIG. 51). The full-length PRO352 protein shown inFIG. 2 has an estimated pI of about 4.62. Analysis of the full-lengthPRO352 sequence evidences the presence of a signal peptide from aboutamino acid 1 to about amino acid 28, a transmembrane domain from aboutamino acid 251 to about amino acid 270, potential N-glycosylation sitesfrom about amino acid 91 to about amino acid 94, about amino acid 104 toabout amino acid 107, about amino acid 189 to about amino acid 192 andabout amino acid 215 to about amino acid 218 and a region havinghomology to immunoglobulins and MHC from about amino acid 217 to aboutamino acid 234. Clone UNQ309 (DNA41386-1316) has been deposited withATCC on Mar. 26, 1998 and is assigned ATCC deposit no. 209703.

Analysis of the amino acid sequence of the full-length PRO352polypeptide suggests that it possesses significant sequence similarityto the butyrophilin protein, thereby indicating that PRO352 is a novelbutyrophilin homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO352 amino acid sequence and the following Dayhoffsequences, BUTY_HUMAN, HSB73_(—)1, GGCD80_(—)1, I46690, A33_HUMAN,P_R67988, CD86_MOUSE, P_R71360, B39371 and D50558_(—)1.

Example 24 Isolation of cDNA Clones Encoding Human PRO381

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA39651. Based on the DNA39651 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO381.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CTTTCCTTGCTTCAGCAACATGAGGC-3′ (SEQ ID NO:146)-   reverse PCR primer 5′-GCCCAGAGCAGGAGGAATGATGAGC-3′ (SEQ ID NO:147)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA39651 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GTGGAACGCGGTCTTGACTCTGTTCGTCACTTTGATTGGGGCTG-3′ (SEQ ID NO:148)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO381 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO381 [herein designated as UNQ322(DNA44194-1317)] (SEQ ID NO:144) and the derived protein sequence forPRO381.

The entire nucleotide sequence of UNQ322 (DNA44194-1317) is shown inFIG. 52 (SEQ ID NO:144). Clone UNQ322 (DNA44194-1317) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 174–176 and ending at the stop codon at nucleotidepositions 807–809 (FIG. 52). The predicted polypeptide precursor is 211amino acids long (FIG. 53). The full-length PRO381 protein shown in FIG.53 has an estimated molecular weight of about 24,172 daltons and a pI ofabout 5.99. Analysis of the full-length PRO381 polypeptide shown in FIG.53 (SEQ ID NO:145) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 20, a potentialN-glycosylation site from about amino acid 176 to about amino acid 179,potential casein kinase II phosphorylation sites from about amino acid143 to about amino acid 146, from about amino acid 156 to about aminoacid 159, from about amino acid 178 to about amino acid 181, and fromabout amino acid 200 to about amino acid 203, an endoplasmic reticulumtargeting sequence from about amino acid 208 to about amino acid 211,FKBP-type peptidyl-prolyl cis-trans isomerase sites from about aminoacid 78 to about amino acid 114 and from about amino acid 118 to aboutamino acid 131, EF-hand calcium binding domains from about amino acid191 to about amino acid 203, from about amino acid 184 to about aminoacid 203 and from about amino acid 140 to about amino acid 159, and anS-100/ICaBP type calcium binding domain from about amino acid 183 toabout amino acid 203. Clone UNQ322 (DNA44194-1317) has been depositedwith ATCC on Apr. 28, 1998 and is assigned ATCC deposit no. 209808.

Analysis of the amino acid sequence of the full-length PRO381polypeptide suggests that it possesses significant sequence similarityto FKBP immunophilin proteins, thereby indicating that PRO381 may be anovel FKBP immunophilin homolog. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO381 amino acid sequence and the followingDayhoff sequences, AF040252_(—)1, I49669, P_R93551, S71238,CELC05C8_(—)1, CEU27353_(—)1, MIP_TRYCR, CEZC455_(—)3, FKB4_HUMAN andI40718.

Example 25 Isolation of cDNA Clones Encoding Human PRO386

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA40674. Two proprietary Genentech EST sequenceswere employed in the consensus sequence assembly, wherein those ESTsequences are herein designated DNA23350 (FIG. 56; SEQ ID NO:151) andDNA23536 (FIG. 57; SEQ ID NO:152). Based on the DNA40674 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO386.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-ACGGAGCATGGAGGTCCACAGTAC-3′ (SEQ ID NO:153)-   reverse PCR primer 5′-GCACGTTTCTCAGCATCACCGAC-3′ (SEQ ID NO:154)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA40674 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CGCCTGCCCTGCACCTTCAACTCCTGCTACACAGTGAACCACAAACAGTT-3′ (SEQ ID    NO:155)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO386 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal brain tissue (LIB153).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO386 herein designated as UNQ326(DNA45415-1318)] (SEQ ID NO:149) and the derived protein sequence forPRO386.

The entire nucleotide sequence of UNQ326 (DNA45415-1318) is shown inFIG. 54 (SEQ ID NO:149). Clone UNQ326 (DNA45415-1318) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 146–148 and ending at the stop codon at nucleotidepositions 791–793 (FIG. 54). The predicted polypeptide precursor is 215amino acids long (FIG. 55). The full-length PRO386 protein shown in FIG.55 has an estimated molecular weight of about 24,326 daltons and a pI ofabout 6.32. Analysis of the full-length PRO386 sequence shown in FIG. 55(SEQ ID NO:150) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 20, a transmembranedomain from about amino acid 161 to about amino acid 179, animmunoglobulin-like fold from about amino acid 83 to about amino acid127 and potential N-glycosylation sites from about amino acid 42 toabout amino acid 45, from about amino acid 66 to about amino acid 69 andfrom about amino acid 74 to about amino acid 77. Clone UNQ326(DNA45415-1318) has been deposited with ATCC on Apr. 28, 1998 and isassigned ATCC deposit no. 209810.

Analysis of the amino acid sequence of the full-length PRO386polypeptide suggests that it possesses significant sequence similarityto the sodium channel beta-2 subunit, thereby indicating that PRO386 isa novel homolog thereof. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO386 amino acid sequence and the following Dayhoffsequences, A57843, MYP0_HUMAN, GEN14531, JC4024, HS46KDA_(—)1,HSU90716_(—)1, D86996_(—)2, MUSIGLVD_(—)1, DMU42768_(—)1 and S19247.

Example 26 Isolation of cDNA Clones Encoding Human PRO540

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA39631. Based on the DNA39631 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO540.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-CTGGGGCTACACACGGGGTGAGG-3′ (SEQ ID NO:158)-   reverse PCR primer 5′-GGTGCCGCTGCAGAAAGTAGAGCG-3′ (SEQ ID NO:159)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA40654 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GCCCCAAATGAAAACGGGCCCTACTTCCTGGCCCTCCGCGAGATG-3′ (SEQ ID NO:160)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO540 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO540 [herein designated as UNQ341(DNA44189-1322)] (SEQ ID NO:156) and the derived protein sequence forPRO540.

The entire nucleotide sequence of UNQ341 (DNA44189-1322) is shown inFIG. 58 (SEQ ID NO:156). Clone UNQ341 (DNA44189-1322) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 21–23 and ending at the stop codon at nucleotidepositions 1257–1259 (FIG. 58). The predicted polypeptide precursor is412 amino acids long (FIG. 59). The full-length PRO540 protein shown inFIG. 59 has an estimated molecular weight of about 46,658 daltons and apI of about 6.65. Important regions of the amino acid sequence of PRO540include the signal peptide, potential N-glycosylation sites, a potentiallipid substrate binding site, a sequence typical of lipases and serineproteins, and a beta-transducin family Trp-Asp repeat. Clone UNQ341(DNA44189-1322) has been deposited with ATCC and is assigned ATCCdeposit no. 209699.

Example 27 Isolation of cDNA Clones Encoding Human PRO615

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42240. Based on the DNA42240 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO615.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-TGGTCTTCGCCTTGATCGTGTTCT-3′ (SEQ ID NO:163)-   forward PCR primer 5′-GTGTACTGAGCGGCGGTTAG-3′ (SEQ ID NO:164)-   reverse PCR primer 5′-CTGAAGGTGATGGCTGCCCTCAC-3′ (SEQ ID NO:165)-   reverse PCR primer 5′-CCAGGAGGCTCATGGGAAAGTCC-3′ (SEQ ID NO:166)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA42240 sequence which had the    following nucleotide sequence:    hybridization probe-   5′-CCACGAGTCTAAGCAGATGTACTGCGTGTTCAACCGCAACGAGGATGCCT-3′ (SEQ ID    NO:167)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO615 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human bone marrow tissue (LIB255).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO615 [herein designated as UNQ352(DNA43304-1323)] (SEQ ID NO:161) and the derived protein sequence forPRO615.

The entire nucleotide sequence of UNQ352 (DNA48304-1323) is shown inFIG. 60 (SEQ ID NO:161). Clone UNQ352 (DNA48304-1323) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 51–53 and ending at the stop codon at nucleotidepositions 723–725 (FIG. 60). The predicted polypeptide precursor is 224amino acids long (FIG. 61). The full-length PRO615 protein shown in FIG.61 has an estimated molecular weight of about 24,810 daltons and a pI ofabout 4.75. Important regions of the amino acid sequence of PRO615include a type II transmembrane domain, corresponding to about aminoacids 24–43, other transmembrane domains, corresponding to about aminoacids 74–90, 108–126, and 145–161, respectively, and a potentialN-glycosylation site, corresponding to about amino acids 97–100. CloneUNQ352 (DNA48304-1323) has been deposited with ATCC and is assigned ATCCdeposit no. 209811.

Example 28 Isolation of cDNA Clones Encoding Human PRO618

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA30900. Based on the DNA30900 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO618.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-TAACAGCTGCCCACTGCTTCCAGG-3′ (SEQ ID NO:171)-   reverse PCR primer 5′-TAATCCAGCAGTGCAGGCCGGG-3′ (SEQ ID NO:172)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA30900 sequence which had the    following nucleotide sequence    hybridization probe-   5′-ATGGCCTCCACGGTGCTGTGGACCGTGTTCCTGGGCAAGGTGTGGCAGAA-3′ (SEQ ID    NO:173)

Screening of the above described library gave rise to the partial cDNAclone designated herein DNA35597 (SEQ ID NO:170). Extension of thissequence using repeated cycles of BLAST and phrap gave rise to anucleotide sequence designated herein as DNA43335. Primers based uponthe DNA43335 consensus sequence were then prepared as follows.

-   forward PCR primer 5′-TGCCTATGCACTGAGGAGGCAGAAG-3′ (SEQ ID NO:174)-   reverse PCR primer 5′-AGGCAGGGACACAGAGTCCATTCAC-3′ (SEQ ID NO:175)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA43335 sequence which had the    following nucleotide sequence    hybridization probe-   5′-AGTATGATTTGCCGTGCACCCAGGGCCAGTGGACGATCCAGAACAGGAGG-3′ (SEQ ID    NO:176)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate full length clones encoding the PRO618 gene using thesecond probe oligonucleotide and one of the second set of PCR primers.RNA for construction of the cDNA libraries was isolated from human fetalliver tissue (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO618 [herein designated as UNQ354(DNA49152-1324)] (SEQ ID NO:168) and the derived protein sequence forPRO618.

The entire nucleotide sequence of UNQ354 (DNA49152-1324) is shown inFIG. 62 (SEQ ID NO:168). Clone UNQ354 (DNA49152-1324) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 73–75 and ending at the stop codon at nucleotidepositions 2479–2481 (FIG. 62). The predicted polypeptide precursor is802 amino acids long (FIG. 63). The full-length PRO618 protein shown inFIG. 63 has an estimated molecular weight of about 88,846 daltons and apI of about 6.41. Important regions of the amino acid sequence of PRO618include type II transmembrane domain, a sequence typical of a protease,trypsin family, histidine active site, multiple N-glycosylation sites,two sequences typical of a Kringle domain, two regions having sequencesimilarity to Kallikrein light chain, and a region having sequencesimilarity to low-density lipoprotein receptor. Clone UNQ354(DNA49152-1324) has been deposited with ATCC and is assigned ATCCdeposit no. 209813.

Example 29 Isolation of cDNA Clones Encoding Human PRO719

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA44851. Based on the DNA44851 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO719.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GTGAGCATGAGCGAGCCGTCCAC-3′ (SEQ ID NO:179)-   reverse PCR primer 5′-GCTATTACAACGGTTCTTGCGGCAGC-3′ (SEQ ID NO:180)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA44851 sequence which had the    following nucleotide sequence    hybridization probe-   5′-TTGACTCTCTGGTGAATCAGGACAAGCCGAGTTTTGCCTTCCAG-3′ (SEQ ID NO:181)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO719 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human placenta tissue (LIB90).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO719 [herein designated as UNQ387(DNA49646-1327)] (SEQ ID NO:177) and the derived protein sequence forPRO719.

The entire nucleotide sequence of UNQ387 (DNA49646-1327) is shown inFIG. 65 (SEQ ID NO:177). Clone UNQ387 (DNA49646-1327) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 223–225 and ending at the stop codon at nucleotidepositions 1285–1287 (FIG. 65). The predicted polypeptide precursor is354 amino acids long (FIG. 66). The full-length PRO719 protein shown inFIG. 66 has an estimated molecular weight of about 39,362 daltons and apI of about 8.35. Analysis of the full length PRO719 sequence evidencesthe presence of a signal peptide from about amino acid 1 to about aminoacid 16 as shown in FIG. 66 (SEQ ID NO:178), a lipase-associatedserine-containing active site at about amino acid 163 to about aminoacid 172, and two potential N-glycosylation sites from about amino acid80 to about amino acid 83 and about amino acid 136 to about amino acid139. Clone UNQ387 (DNA49646-1327) has been deposited with ATCC on Mar.26, 1998 and is assigned ATCC deposit no. 209705.

Analysis of the amino acid sequence of the full-length PRO719polypeptide suggests that it possesses significant sequence similarityto the lipoprotein lipase H protein, thereby indicating that PRO719 maybe a novel lipoprotein lipase homolog. More specifically, an analysis ofthe Dayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO719 amino acid sequence and the followingDayhoff sequences, LIPL_HUMAN, LIPH_HUMAN, D83548_(—)1, A24059_(—)1,P_R30740, D88666_(—)1, A43357, A46696, B43357 and A49488.

Example 30 Isolation of cDNA Clones Encoding Human PRO724

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA35603. Based on the DNA35603 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO724.

Pairs of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 5′-GGCTGTCACTGTGGAGACAC-3′ (SEQ ID NO:184)-   forward PCR primer 2 5′-GCAAGGTCATTACAGCTG-3′ (SEQ ID NO:185)-   reverse PCR primer 1 5′-AGAACATAGGAGCAGTCCCACTC-3′ (SEQ ID NO:186)-   reverse PCR primer 2 5′-TGCCTGCTGCTGCACAATCTCAG-3′ (SEQ ID NO:187)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA35603 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GGCTATTGCTTGCCTTGGGACAGACCCTGTGGCTTAGGCTCTGGC-3′ (SEQ ID NO:188)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO724 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue (LIB26).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO724 [herein designated as UNQ389(DNA49631-1328)] (SEQ ID NO:182) and the derived protein sequence forPRO724.

The entire nucleotide sequence of UNQ389 (DNA49631-1328) is shown inFIG. 67 (SEQ ID NO:182). Clone UNQ389 (DNA49631-1328) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 546–548 and ending at the stop codon at nucleotidepositions 2685–2687 (FIG. 67). The predicted polypeptide precursor is713 amino acids long (FIG. 68). The full-length PRO724 protein shown inFIG. 68 has an estimated molecular weight of about 76,193 daltons and apI of about 5.42. Analysis of the full-length PRO724 amino acid sequenceshown in FIG. 68 (SEQ ID NO:183) evidences the presence of thefollowing: a signal peptide from about amino acid 1 to about amino acid16, a transmembrane domain from about amino acid 442 to about amino acid462 and LDL receptor class A domain regions from about amino acid 152 toabout amino acid 171, about amino acid 331 to about amino acid 350,about amino acid 374 to about amino acid 393 and about amino acid 411 toabout amino acid 430. Clone UNQ389 (DNA49631-1328) has been depositedwith ATCC on Apr. 28, 1998 and is assigned ATCC deposit no. 209806

Analysis of the amino acid sequence of the full-length PRO724polypeptide suggests that it possesses significant sequence similarityto the human LDL receptor protein, thereby indicating that PRO724 may bea novel LDL receptor homolog. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO724 amino acid sequence and the followingDayhoff sequences, P_R48547, MMAM2R_(—)1, LRP2_RAT, P_R60517, P_R47861,P_R05533, A44513_(—)1, A30363, P_R74692 and LMLIPOPHO_(—)1.

Example 31 Isolation of cDNA Clones Encoding Human PRO772

One cDNA sequence was isolated in the amylase screen described inExample 2, wherein that cDNA sequence is herein designated DNA43509 (seeFIG. 71). Based on the DNA43509 sequence, oligonucleotide probes weregenerated and used to screen a human fetal lung library (LIB25) preparedas described in paragraph 1 of Example 2 above. The cloning vector waspRK5B (pRK5B is a precursor of pRK5D that does not contain the SfiIsite; see, Holmes et al., Science, 253:1278–1280 (1991)), and the cDNAsize cut was less than 2800 bp.

A pair of PCR primers (forward and reverse) were synthesized based onthe DNA43509 sequence:

-   forward PCR primer 5′-CGTTTTGCAGAACCTACTCAGGCAG-3′ (SEQ ID NO:192)-   reverse PCR primer 5′-CCTCCACCAACTGTCAATGTTGTGG-3′ (SEQ ID NO:193)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA43509 sequence which had the    following nucleotide sequence    hybridization probe-   5′-AAAGTGCTGCTGCTGGGTCTGCAGACGCGATGGATAACGT-3′ (SEQ ID NO:194)

Using the above described primers and library, a full length clone wasidentified that contained a single open reading frame with an apparenttranslational initiation site at nucleotide positions 131–133 and endingat the stop codon found at nucleotide positions 587–589 (FIG. 69; SEQ IDNO:189). The predicted polypeptide precursor is 152 amino acids long,has a calculated molecular weight of approximately 17,170 daltons and anestimated pI of approximately 9.62. Analysis of the full-length PRO772sequence shown in FIG. 70 (SEQ ID NO:190) evidences the presence of thefollowing: a potential type II transmembrane domain from about aminoacid 26 to about amino acid 42, other potential transmembrane domainsfrom about amino acid 44 to about amino acid 65, from about amino acid81 to about amino acid 101 and from about amino acid 109 to about aminoacid 129, leucine zipper pattern sequences from about amino acid 78 toabout amino acid 99 and from about amino 7 acid 85 to about amino acid106. Clone UNQ410 (DNA49645-1347) has been deposited with ATCC on Apr.28, 1998 and is assigned ATCC deposit no. 209809.

Analysis of the amino acid sequence of the full-length PRO772polypeptide suggests that it possesses significant sequence similarityto the human A4 protein, thereby indicating that PRO772 may be a novelA4 protein homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO772 amino acid sequence and the following Dayhoffsequences, HSU93305_(—)1, A4P_HUMAN, CELB0454_(—)2, VPU_JSRV,CELC12D12_(—)2, OCCM_AGRT1, LBPHIG1E_(—)50, YIGK_(—) ECOLI, S76245 andP_R50807.

Example 32 Isolation of cDNA Clones Encoding Human PRO852

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA34364. Based on the DNA34364 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO852.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 5′-CGCAGAAGCTACAGATTCTCG-3′ (SEQ ID NO:197)-   forward PCR primer 2 5′-GGAAATTGGAGGCCAAAGC-3′ (SEQ ID NO:198)-   forward PCR primer 3 5′-GGATGTAGCCAGCAACTGTG-3′ (SEQ ID NO:199)-   forward PCR primer 4 5′-GCCTTGGCTCGTTCTCTTC-3′ (SEQ ID NO:200)-   forward PCR primer 5 5′-GGTCCTGTGCCTGGATGG-3′ (SEQ ID NO:201)-   reverse PCR primer 1 5′-GACAAGACTACCTCCGTTGGTC-3′ (SEQ ID NO:202)-   reverse PCR primer 2 5′-TGATGCACAGTTCAGCACCTGTTG-3′ (SEQ ID NO:203)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA34364 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CGCTCCAAGGGCTTTGACGTCACAGTGAAGTACACACAAGGAAGCTG-3′ (SEQ ID    NO:204)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO852 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB228).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO852 [herein designated as UNQ418(DNA45493-1349)] (SEQ ID NO:195) and the derived protein sequence forPRO852.

The entire nucleotide sequence of UNQ418 (DNA45493-1349) is shown inFIG. 72 (SEQ ID NO:195). Clone UNQ418 (DNA45493-1349) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 94–96 and ending at the stop codon at nucleotidepositions 16748–1650 (FIG. 72). The predicted polypeptide precursor is518 amino acids long (FIG. 73). The full-length PRO852 protein shown inFIG. 73 has an estimated molecular weight of about 56,180 daltons and apI of about 5.08. Analysis of the full-length PRO852 sequence shown inFIG. 73 (SEQ ID NO:196) evidences the presence of the following: asignal peptide from about amino acid 1 to about amino acid 20, atransmembrane domain from about amino acid 466 to about amino acid 494,potential N-glycosylation sites from about amino acid 170 to about aminoacid 173 and about amino acid 366 to about amino acid 369, leucinezipper sequence pattern blocks from about amino acid 10 to about aminoacid 31 and from about amino acid 197 to about amino acid 218 and blocksof amino acids having sequence homology to eukaryotic and viral aspartylproteases from about amino acid 109 to about amino acid 118, from aboutamino acid 252 to about amino acid 261 and from about amino acid 298 toabout amino acid 310. Clone UNQ418 (DNA45493-1349) has been depositedwith ATCC on Apr. 28, 1998 and is assigned ATCC deposit no. 209805.

Analysis of the amino acid sequence of the full-length PRO852polypeptide suggests that it possesses significant sequence similarityto various protease proteins, thereby indicating that PRO852 may be anovel protease protein or homolog thereof. More specifically, ananalysis of the Dayhoff database (version 35.45 SwissProt 35) evidencedsignificant homology between the PRO852 amino acid sequence and thefollowing Dayhoff sequences, PEPC_HUMAN, S66516, S66517, PEPE_CHICK,CATD^(—)HUMAN, P_R74207, CARP_YEAST, PEP2_RABIT, CATE_HUMAN andRENI_MOUSE.

Example 33 Isolation of cDNA Clones Encoding Human PRO853

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43050. Based on the DNA43050 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO853.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-CTTCATGGCCTTGGACTTGGCCAG-3′ (SEQ ID NO:207)-   reverse PCR primer 5′-ACGCCAGTGGCCTCAAGCTGGTTG-3′ (SEQ ID NO:208)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA43050 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CTTTCTGAGCTCTGAGCCACGGTTGGACATCCTCATCCACAATGC-3′ (SEQ ID NO:209)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO853 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB228).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO853 [herein designated as UNQ419(DNA48227-1350)] (SEQ 1D NO:205) and the derived protein sequence forPRO853.

The entire nucleotide sequence of UNQ419 (DNA48227-1350) is shown inFIG. 74 (SEQ ID NO:205). Clone UNQ419 (DNA48227-1350) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 128–130 and ending at the stop codon at nucleotidepositions 1259–1261 (FIG. 74). The predicted polypeptide precursor is377 amino acids long (FIG. 75). The full-length PRO853 protein shown inFIG. 75 has an estimated molecular weight of about 40,849 daltons and api of about 7.98. Important regions of the amino acid sequence of PRO853include the signal peptide, corresponding to amino acids from about 1 toabout 16 of SEQ ID NO:206, the glycosaminoglycan attachment site,corresponding to amino acids from about 46 to about 49 of SEQ ID NO:206,and two sequences typical of the short-chain alcohol dehydrogenasefamily, corresponding to amino acids from about 37 to about 49 and about114 to about 124 of SEQ ID NO:206, respectively. Clone UNQ419(DNA48227-1350) has been deposited with ATCC and is assigned ATCCdeposit no. 209812.

Example 34 Isolation of cDNA Clones Encoding Human PRO860

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA38137. Based on the DNA38137 consensu sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO860.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-GAAGGGACCTACATGTGTGTGGCC-3′ (SEQ ID NO:212)-   reverse PCR primer 5′-ACTGACCTTCCAGCTGAGCCACAC-3′ (SEQ ID NO:213)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA40654 sequence which had the    following nucleotide sequence    hybridization probe-   5′-AGGACTACACGGAGCCTGTGGAGCTTCTGGCTGTGCGAATTCAGCTGGAA-3′ (SEQ ID    NO:214)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO860 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal lung tissue (LIB26).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO860 [herein designated as UNQ421(DNA41404-1352)] (SEQ ID NO:210) and the derived protein sequence forPRO860.

The entire nucleotide sequence of UNQ421 (DNA41404-1352) is shown inFIG. 76 (SEQ ID NO:210). Clone UNQ421 (DNA41404-1352) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 58–60 and ending at the stop codon at nucleotidepositions 3013–3015 (FIG. 76). The predicted polypeptide precursor is985 amino acids long (FIG. 77). The full-length PRO860 protein shown inFIG. 77 has an estimated molecular weight of about 105,336 daltons and apI of about 6.55. Important regions of the amino acid sequence of PRO860include the transmembrane region corresponding to about amino acids448–467, the extracellular domain, corresponding to amino acids about1–447, several N-glycosylation sites, numerous N-myristoylation sitesand a sequence typical of phosphotyrosine interaction domain proteins.Clone UNQ421 (DNA41404-1352) has been deposited with ATCC and isassigned ATCC deposit no. 209844.

Example 35 Isolation of cDNA Clones Encoding Human PRO846

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA39949. Based on the DNA39949 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO846.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-CCCTGCAGTGCACCTACAGGGAAG-3′ (SEQ ID NO:217)-   reverse PCR primer 5′-CTGTCTTCCCCTGCTTGGCTGTGG-3′ (SEQ ID NO:218)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA39949 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GGTGCAGGAAGGGTGGGATCCTCTTCTCTCGCTGCTCTGGCCACATC-3′ (SEQ ID    NO:219)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO546 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO846 [herein designated as UNQ422(DNA44196-1353)] (SEQ ID NO:215) and the derived protein sequence forPRO846.

The entire nucleotide sequence of UNQ422 (DNA44196-1353) is shown inFIG. 78 (SEQ ID NO:215). Clone UNQ422 (DNA44196-1353) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 25–27 and ending at the stop codon at nucleotidepositions 1021–1023 (FIG. 78). The predicted polypeptide precursor is332 amino acids long (FIG. 79). The full-length PRO846 protein shown inFIG. 79 has an estimated molecular weight of about 36,143 daltons and apI of about 5.89. Important regions of the amino acid sequence of PRO846include the signal peptide, the transmembrane domain, an N-glycosylationsite, a sequence typical of fibrinogen beta and gamma chains C-terminaldomain, and a sequence typical of Ig like V-type domain as shown in FIG.79. Clone UNQ422 (DNA44196-1353) has been deposited with ATCC and isassigned ATCC deposit no. 209847.

Example 36 Isolation of cDNA Clones Encoding Human PRO862

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA47370. Based on the DNA47370 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO862.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-GCTGCAGCTGCAAATTCCACTGG-3′ (SEQ ID NO:227)-   reverse PCR primer 5′-TGGTGGGAGACTGTTTAAATTATCGGCC-3′ (SEQ ID    NO:228)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA47370 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CTGCCTGCTACCCTCCAAGTGAGGCCAAGCTCTACGGTCGTTGTG-3′ (SEQ ID NO:225)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO862 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human pancreas tissue (LIB55).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO862 [herein designated as UNQ424(DNA52187-1354)] (SEQ ID NO:220) and the derived protein sequence forPRO862.

The entire nucleotide sequence of UNQ424 (DNA52187-1354) is shown inFIG. 80 (SEQ ID NO:220). Clone UNQ424 (DNA52187-1354) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 410–412 and ending at the stop codon at nucleotidepositions 848–850 (FIG. 80). The predicted polypeptide precursor is 146amino acids long (FIG. 81). The full-length PRO862 protein shown in FIG.81 has an estimated molecular weight of about 16,430 daltons and a pI ofabout 5.05. Important regions of the amino acid sequence of PRO862include the signal peptide, an N-myristoylation site, and sequenceshaving similarity to region to Alpha-lactalbumin/lysozyme C proteins asshown in FIG. 81. Clone UNQ424 (DNA52187-1354) has been deposited withthe ATCC and is assigned ATCC deposit no. 209845.

Example 37 Isolation of cDNA Clones Encoding Human PRO864

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA40666. Based on the DNA40666 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO864.

Forward and reverse PCR primers were synthesized:

-   forward PCR primer 5′-GCTGCAGCTGCAAATTCCACTGG-3′ (SEQ ID NO:227)-   reverse PCR primer 5′-TGGTGGGAGACTGTTTAAATTATCGGCC-3′ (SEQ ID    NO:228)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA40666 sequence which had the    following nucleotide sequence    hybridization probe-   5′-TGCTTCGTCAAGTGCCGGCAGTGCCAGCGGCrCGTGGAGTT-3′ (SEQ ID NO:229)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO864 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal brain tissue (LIB153).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO864 [herein designated as UNQ426(DNA48328-1355)] (SEQ ID NO:225) and the derived protein sequence forPRO864.

The entire nucleotide sequence of UNQ426 (DNA48328-1355) is shown inFIG. 82 (SEQ ID NO:225). Clone UNQ426 (DNA48328-1355) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 37–39 and ending at the stop codon at nucleotidepositions 1090–1092 (FIG. 82). The predicted polypeptide precursor is351 amino acids long (FIG. 83). The full-length PRO864 protein shown inFIG. 83 has an estimated molecular weight of about 39,052 and a pI ofabout 8.97. Important regions of the amino acid sequence of PRO864include the signal peptide, two N-glycosylation sites, a Wnt-1 familysignature sequence, and sequence regions homologous to Wnt-1 familyproteins as shown in FIG. 83. Clone UNQ426 (DNA48328-1355) has beendeposited with ATCC and is assigned ATCC deposit no. 209843.

Example 38 Isolation of cDNA Clones Encoding Human PRO792

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA38106. Based on the DNA38106 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO792.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GCGAGAACTGTGTCATGATGCTGC-3′ (SEQ ID NO:232)-   reverse PCR primer 5′-GTTTCTGAGACTCAGCAGCGGTGG-3′ (SEQ ID NO:233)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA38106 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CACCGTGTGACAGCGAGAAGGACGGCTGGATCTGTGAGAAAAGGCACAAC-3′ (SEQ ID    NO:234)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO792 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human bone marrow tissue (LIB255).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO792 [herein designated as UNQ431(DNA56352-1358)] (SEQ ID NO:230) and the derived protein sequence forPRO792.

The entire nucleotide sequence of UNQ431 (DNA56352-1358) is shown inFIG. 84 (SEQ ID NO:230). Clone UNQ431 (DNA56352-1358) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 67–69 and ending at the stop codon at nucleotidepositions 946–948 (FIG. 84). The predicted polypeptide precursor is 293amino acids long (FIG. 85). The full-length PRO792 protein shown in FIG.85 has an estimated molecular weight of about 32,562 daltons and a pI ofabout 6.53. Analysis of the full-length PRO792 sequence shown in FIG. 85(SEQ ID NO:231) evidences the presence of the following: a type IItransmembrane domain from about amino acid 31 to about amino acid 54,potential N-glycosylation sites from about amino acid 73 to about aminoacid 76 and from about amino acid 159 to about amino acid 162, a leucinezipper amino acid sequence pattern from about amino acid 102 to aboutamino acid 123, potential N-myristolation sites from about amino acid 18to about amino acid 23, from about amino acid 133 to about amino acid138 and from about amino acid 242 to about amino acid 247 and a C-typelectin domain signature block from about amino acid 264 to about aminoacid 287. Clone UNQ431 (DNA56352-1358) has been deposited with ATCC onMay 6, 1998 and is assigned ATCC deposit no. 209846.

Analysis of the amino acid sequence of the full-length PRO792polypeptide suggests that it possesses significant sequence similarityto the CD23 protein, thereby indicating that PRO792 may be a novel CD23homolog. More specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35) evidenced significant homology between the PRO792amino acid sequence and the following Dayhoff sequences, S34198,A07100_(—)1, A05303_(—)1, P_R41689, P_P82839, A10871_(—)1, P_R12796,P_R47199, A46274 and P_R32188.

Example 39 Isolation of cDNA Clones Encoding Human PRO866

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA44708. Based on the DNA44708 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO866.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 5′-CAGCACTGCCAGGGGAAGAGGG-3′ (SEQ ID NO:237)-   forward PCR primer 2 5′-CAGGACTCGCTACGTCCG-3′ (SEQ ID NO:238)-   forward PCR Primer 3 5′-CAGCCCCTTCTCCTCCTTTCTCCC-3′ (SEQ ID NO:239)-   reverse PCR Primer 1 5′-GCAGTTATCAGGGACGCACTCAGCC-3′ (SEQ ID NO:240)-   reverse PCR primer 2 5′-CCAGCGAGAGGCAGATAG-3′ (SEQ ID NO:241)-   reverse PCR primer 3 5′-CGGTCACCGTGTCCTGCGGGATG-3′ (SEQ ID NO:242)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA44708 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CAGCCCCTTCTCCTCCTTTCTCCCACGTCCTATCTGCCTCTC-3′ (SEQ ID NO:243)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO866 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB228).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO866 [herein designated as UNQ435(DNA53971-1359)] (SEQ ID NO:235) and the derived protein sequence forPRO866.

The entire nucleotide sequence of UNQ435 (DNA53971-1359) is shown inFIG. 86 (SEQ ID NO:235). Clone UNQ435 (DNA53971-1359) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 275–277 and ending at the stop codon at nucleotidepositions 1268–1270 (FIG. 86). The predicted polypeptide precursor is331 amino acids long (FIG. 87). The full-length PRO866 protein shown inFIG. 87 has an estimated molecular weight of about 35,844 daltons and apI of about 5.45. Analysis of the full-length PRO866 sequence shown inFIG. 87 (SEQ ID NO:236) evidences the presence of the following: asignal peptide from about amino acid 1 to about amino acid 26. CloneUNQ435 (DNA53971-1359) has been deposited with ATCC on Apr. 7, 1998 andis assigned ATCC deposit no. 209750.

Analysis of the amino acid sequence of the full-length PRO866polypeptide suggests that it possesses significant sequence similarityto the mindin/spondin family of proteins, thereby indicating that PRO866may be a novel mindin homolog. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO866 amino acid sequence and the followingDayhoff sequences, AB006085_(—)1, AB006084_(—)1, AB006086_(—)1,AF017267_(—)1, CWU42213_(—)1, AC004160_(—)1, CPMICRP1, S49108, A48569and I46687.

Example 40 Isolation of cDNA Clones Encoding Human PRO871

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA40324. Based on the DNA40324 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO871.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 5′-TGCGGAGATCCTACTGGCACAGGG-3′ (SEQ ID NO:246)-   forward PCR primer 2 5′-CGAGTTAGTCAGAGCATG-3′ (SEQ I) NO:247)-   forward PCR Primer 3 5′-CAGATGGTGCTGTTGCCG-3′ (SEQ ID NO:248)-   reverse PCR primer 1 5′-CAACTGGAACAGGAACTGAGATGTGGATC-3′ (SEQ ID    NO:249)-   reverse PCR primer 2 5′-CTGGTTCAGCAGTGCAAGGGTCTG-3′ (SEQ ID NO:250)-   reverse PCR primer 3 5′-CCTCTCCGATTAAAACGC-3′ (SEQ ID NO:251)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA40324 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GAGAGGACTGGTTGCCATGGCAAATGCTGGTTCTCATGATAATGG-3′ (SEQ ID NO:252)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO871 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO871 [herein designated as UNQ438(DNA50919-1361)] (SEQ ID NO:244) and the derived protein sequence forPRO871.

The entire nucleotide sequence of UNQ438 (DNA50919-1361) is shown inFIG. 88 (SEQ ID NO:244). Clone UNQ438 (DNA50919-1361) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 191–193 and ending at the stop codon at nucleotidepositions 1607–1609 (FIG. 88). The predicted polypeptide precursor is472 amino acids long (FIG. 89). The full-length PRO871 protein shown inFIG. 89 has an estimated molecular weight of about 53,847 daltons and apI of about 5.75. Analysis of the full-length PRO871 sequence shown inFIG. 89 (SEQ ID NO:245) evidences the presence of the following: asignal peptide from about amino acid 1 to about amino acid 21, potentialN-glycosylation sites from about amino acid 109 to about amino acid 112and from about amino acid 201 to about amino acid 204, acyclophilin-type peptidy-prolyl cis-trans isomerase signature sequencefrom about amino acid 49 to about amino acid 66 and regions that arehomologous to cyclophilin-type peptidy-prolyl cis-trans isomerases fromabout amino acid 96 to about amino acid 140, from about amino acid 49 toabout amino acid 89 and from about amino acid 22 to about amino acid 51.Clone UNQ438 (DNA50919-1361) has been deposited with ATCC on May 6, 1998and is assigned ATCC deposit no. 209848.

Analysis of the amino acid sequence of the full-length PRO871polypeptide suggests that it possesses significant sequence similarityto the cyclophilin family of proteins, thereby indicating that PRO871may be a novel cyclophilin protein family member. More specifically, ananalysis of the Dayhoff database (version 35.45 SwissProt 35) evidencedsignificant homology between the PRO871 amino acid sequence and thefollowing Dayhoff sequences, SPBC16H5_(—)5, S64705, YAL5_SCHPO,CYP4_CAEEL, CELC34D4_(—)7, CYPA_CAEEL, HUMORF006_(—)1, CYPI_MYCTU,AF043642_(—)1 and HSSRCYP_(—)1.

Example 41 Isolation of cDNA Clones Encoding Human PRO873

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA39621. Based on the DNA39621 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO873.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-AGGTGCCTGCAGGAGTCCTGGGG-3′ (SEQ ID NO:255)-   reverse PCR primer 5′- CCACCTCAGGAAGCCGAAGATGCC-3′ (SEQ ID NO:256)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA39621 sequence which had the    following nucleotide sequence:    hybridization probe-   5′-GAACGGTACAAGTGGCTGCGCTTCAGCGAGGACTGTCTGTACCTG-3′ (SEQ ID NO:257)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO873 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO873 [herein designated as UNQ440(DNA44179-1362)] (SEQ ID NO:253) and the derived protein sequence forPRO873.

The entire nucleotide sequence of UNQ440 (DNA44179-1362) is shown inFIG. 90 (SEQ ID NO:253). Clone UNQ440 (DNA44179-1362) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 139–141 and ending at the stop codon at nucleotidepositions 1774–1776 (FIG. 90). The predicted polypeptide precursor is545 amino acids long (FIG. 91). The full-length PRO873 protein shown inFIG. 91 has an estimated molecular weight of about 58,934 daltons and apI of about 9.45. Analysis of the full-length PRO873 sequence shown inFIG. 91 (SEQ ID NO:254) evidences the presence of the followingfeatures: a signal peptide from about amino acid 1 to about amino acid29; a carboxylesterase type-B serine active site at about amino acid 312to about amino acid 327; a carboxylesterase type-B signature 2 motif atabout amino acid 218 to about amino acid 228; and three potentialN-glycosylation sites at about amino acid 318 to about amino acid 321,about amino acid 380 to about amino acid 383, and about amino acid 465to about amino acid 468. Clone UNQ440 (DNA44179-1362) has been depositedwith ATCC on May 6, 1998 and is assigned ATCC deposit no. 209851.

Analysis of the amino acid sequence of the full-length PRO873polypeptide suggests that it possesses significant sequence similarityto a human liver carboxylesterase, thereby indicating that PRO873 may bea novel carboxylesterase. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO873 amino acid sequence and the following Dayhoffsequences: ES10_RAT, GEN12405, AB010633_(—)1, EST4_RAT, A48809,SASB_ANAPL, RNU41662_(—)1, RNU22952_(—)1, BAL_RAT, GEN13522.

Example 42 Isolation of cDNA Clones Encoding Human PRO940

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA47442. Based on the DNA47442 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO940.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR Primer 5′-CAAAGCCTGCGCCTGGTCTGTG-3′ (SEQ ID NO:260)-   reverse PCR primer 5′-TTCTGGAGCCCAGAGGGTGCTGAG-3′ (SEQ ID NO:262)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA47442 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GGAGCTGCCACCCATTCAAATGGAGCACGAAGGAGAGTTCACCTG-3′ (SEQ ID NO:263)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was ii screened by PCR amplification withthe PCR primer pair identified above. A positive library was then usedto isolate clones encoding the PRO940 gene using the probeoligonucleotide and one of the PCR primers. RNA e for construction ofthe cDNA libraries was isolated from human fetal liver tissue (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO940 [herein designated as UNQ477(DNA54002-1367)] (SEQ ID NO:258) and the derived protein sequence forPRO940.

The entire nucleotide sequence of UNQ477 (DNA54002-1367) is shown inFIG. 92 (SEQ ID NO:258). Clone UNQ477 (DNA54002-1367) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 46–48 and ending at the stop codon at nucleotidepositions 1678–1680 (FIG. 92). The predicted polypeptide precursor is544 amino acids long (FIG. 93). The full-length PRO940 protein shown inFIG. 93 has an estimated molecular weight of about 60,268 daltons and apI of about 9.53. Analysis of the full-length PRO940 sequence shown inFIG. 93 (SEQ ID NO:259) evidences the presence of the following: asignal peptide from about amino acid 1 to about amino acid 15, potentialN-glycosylation sites from about amino acid 100 to about amino acid 103,from about amino acid 297 to about amino acid 300 and from about aminoacid 306 to about amino acid 309 and an immunoglobulin and majorhistocompatibility complex signature sequence block from about aminoacid 365 to about amino acid 371. Clone UNQ477 (DNA54002-1367) has beendeposited with ATCC on Apr. 7, 1998 and is assigned ATCC deposit no.209754.

Analysis of the amino acid sequence of the full-length PRO940polypeptide suggests that it possesses significant sequence similarityto CD33 and the OB binding protein-2. More specifically, an analysis ofthe Dayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO940 amino acid sequence and the followingDayhoff sequences, CD33_HUMAN, HSU71382_(—)1, HSU71383_(—)1,D86359_(—)1, PGBM_HUMAN, MAGS_MOUSE, D86983_(—)1, C22B_HUMAN, P_W01002and HVU24116_(—)1.

Example 43 Isolation of cDNA Clones Encoding Human PRO941

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA35941. An EST sequence proprietary to Genentechwas employed in the assembly and is herein designated DNA6415 (FIG. 96;SEQ ID NO:265). Based on the DNA35941 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO941.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CTTGACTGTCTCTGAATCTGCACCC-3′ (SEQ ID NO:266)-   reverse PCR primer 5′-AAGTGGTGGAAGCCTCCAGTGTGG-3′ (SEQ ID NO:267)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA35941 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CCACTACGGTATTAGAGCAAAAGTTAAAACCATCATGGTTCCTGGAGCAGC-3′ (SEQ ID    NO:268)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO941 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO941 [herein designated as UNQ478(DNA53906-1368)] (SEQ ID NO:263) and the derived protein sequence forPRO941.

The entire nucleotide sequence of UNQ478 (DNA53906-1368) is shown inFIG. 94 (SEQ ID NO:263). Clone UNQ478 (DNA53906-1368) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 37–39 and ending at the stop codon at nucleotidepositions 2353–2355 (FIG. 94). The predicted polypeptide precursor is772 amino acids long (FIG. 95). The full-length PRO941 protein shown inFIG. 95 has an estimated molecular weight of about 87,002 daltons and apI of about 4.64. Analysis of the full-length PRO941 sequence shown inFIG. 95 (SEQ ID NO:264) evidences the presence of the following: asignal peptide from about amino acid 1 to about amino acid 21, potentialN-glycosylation sites from about amino acid 57 to about amino acid 60,from about amino acid 74 to about amino acid 77, from about amino acid419 to about amino acid 422, from about amino acid 437 to about aminoacid 440, from about amino acid 508 to about amino acid 511, from aboutamino acid 515 to about amino acid 518, from about amino acid 516 toabout amino acid 519 and from about amino acid 534 to about amino acid537, and cadherin extracellular repeated domain signature sequences fromabout amino acid 136 to about amino acid 146 and from about amino acid244 to about amino acid 254. Clone UNQ478 (DNA53906-1368) has beendeposited with ATCC on Apr. 7, 1998 and is assigned ATCC deposit no.209747.

Analysis of the amino acid sequence of the full-length PRO941polypeptide suggests that it possesses significant sequence similarityto a cadherin protein, thereby indicating that PRO941 may be a novelcadherin protein family member. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO941 amino acid sequence and the followingDayhoff sequences, I50180, CADA_CHICK, I50178, GEN12782, CADC_HUMAN,P_W25637, A38992, P_R49731, D38992 and G02678.

Example 44 Isolation of cDNA Clones Encoding Human PRO944

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA47374. A variety of proprietary Genentech ESTsequences were employed in the assembly and are shown in FIGS. 99–107.Based on the DNA47374 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO944.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CGAGCGAGTCATGGCCAACGC-3′ (SEQ ID NO:280)-   reverse PCR primer 5′-GTGTCACACGTAGTCTTTCCCGCTGG-3′ (SEQ ID NO:281)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA47374 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CTGCAGCTGTTGGGCTTCATTCTCGCCTTCCTGGGATGGATCG-3′ (SEQ ID NO:282)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO944 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO944 [herein designated as UNQ481(DNA52185-1370)] (SEQ ID NO:269) and the derived protein sequence forPRO944.

The entire nucleotide sequence of UNQ481 (DNA52185-1370) is shown inFIG. 97 (SEQ ID NO:269). Clone UNQ481 (DNA52185-1370) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 219–221 and ending at the stop codon at nucleotidepositions 852–854 (FIG. 97). The predicted polypeptide precursor is 211amino acids long (FIG. 98). The full-length PRO944 protein shown in FIG.98 has an estimated molecular weight of about 22,744 daltons and a pI ofabout 8.51. Analysis of the full-length PRO944 sequence shown in FIG. 98(SEQ ID NO:270) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 21, transmembranedomains from about amino acid 82 to about amino acid 102, from aboutamino acid 118 to about amino acid 142 and from about amino acid 161 toabout amino acid 187, a potential N-glycosylation site from about aminoacid 72 to about amino acid 757 a sequence block having homology toPMP-22/EMP/MP20 family of proteins from about amino acid 70 to aboutamino acid 111 and a sequence block having homology to ABC-2 typetransport system integral membrane protein from about amino acid 119 toabout amino acid 133. Clone UNQ481 (DNA52185-1370) has been depositedwith ATCC on May 14, 1998 and is assigned ATCC deposit no. 209861.

Analysis of the amino acid sequence of the full-length PRO944polypeptide suggests that it possesses significant sequence similarityto the CPE-R protein, thereby indicating that PRO944 may be a novelCPE-R homolog. More specifically, an analysis of the Dayhoff database(version 35.45 SwissProt 35) evidenced significant homology between thePRO944 amino acid sequence and the following Dayhoff sequences,AB000713_(—)1, AB000714_(—)1, AF035814_(—)1, AF000959_(—)1,HSU89916_(—)1, EMP2_HUMAN, JC5732, CELF53B3_(—)6, PM22_MOUSE andCGU49797_(—)1.

Example 45 Isolation of cDNA Clones Encoding Human PRO983

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA47473. Various proprietary Genentech ESTsequences were employed in the assembly, wherein those EST sequences areshown in FIGS. 110–116. Based on the DNA47473 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO983.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GCACCACCGTAGGTACTTGTGTGAGGC-3′ (SEQ ID NO:292)-   reverse PCR primer 5′-AACCACCAGAGCCAAGAGCCGGG-3′ (SEQ ID NO:293)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA47473 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CAGCGGAATCATCGATGCAGGGGCCTCAATTAATGTATCTGTGATGTTAC-3′ (SEQ ID    NO:294)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO983 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human bone marrow (LIB256).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO983 [herein designated as UNQ484(DNA53977-1371)] (SEQ ID NO:283) and the derived protein sequence forPRO983.

The entire nucleotide sequence of UNQ484 (DNA53977-1371) is shown inFIG. 108 (SEQ ID NO:283). Clone UNQ484 (DNA53977-1371) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 234–236 and ending at the stop codon at nucleotidepositions 963–965 (FIG. 108). The predicted polypeptide precursor is 243amino acids long (FIG. 109). The full-length PRO983 protein shown inFIG. 109 has an estimated molecular weight of about 27,228 daltons and apI of about 7.43. Analysis of the full-length PRO983 sequence shown inFIG. 109 (SEQ ID NO:284) evidences the presence of the followingfeatures: a putative transmembrane domain from about amino acid 224 toabout amino acid 239; a potential N-glycosylation site from about aminoacid 68 to about amino acid 71; and three potential N-myristoylationsites from about amino acid 59 to about amino acid 64, from about aminoacid 64 to about amino acid 69, and from about amino acid 235 to aboutamino acid 240. Clone UNQ484 (DNA53977-1371) has been deposited withATCC on May 14, 1998 and is assigned ATCC deposit no. 209862.

Analysis of the amino acid sequence of the full-length PRO983polypeptide suggests that it possesses significant sequence similarityto the vesicle-associated protein, VAP-33, thereby indicating thatPRO983 may be a novel vesicle associated membrane protein. Morespecifically, an analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced significant homology between the PRO983 aminoacid sequence and the following Dayhoff sequences: VP33_APLCA,CELF33D11_(—)12, CELF42G2_(—)2, S50623, YDFC_SCHPO, CELF54H5_(—)2,CELZC196_(—)8, CEF57A10_(—)3, MSP3_GLORO, CEC15H1_(—)1.

Example 46 Isolation of cDNA Clones Encoding Human PRO1057

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA49808. Based on the DNA49808 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1057.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GCATCTGCAGGAGAGAGCGAAGGG-3′ (SEQ ID NO:297)-   reverse PCR primer 5′-CATCGTTCCCGTGAATCCAGAGGC-3′ (SEQ ID NO:298)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA49808 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GAAGGGAGGCCTTCCTTTCAGTGGACCCGGGTCAAGAATACCCAC-3′ (SEQ ID NO:299)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1057 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1057 [herein designated as UNQ522(DNA57253-1382)] (SEQ ID NO:295) and the derived protein sequence forPRO1057.

The entire nucleotide sequence of UNQ522 (DNA57253-1382) is shown inFIG. 117 (SEQ ID NO:295). Clone UNQ522 (DNA57253-1382) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 275–277 and ending at the stop codon at nucleotidepositions 1514–1516 (FIG. 117). The predicted polypeptide precursor is413 amino acids long (FIG. 118). The full-length PRO1057 protein shownin FIG. 118 has an estimated molecular weight of about 47,070 daltonsand a pI of about 9.92. Analysis of the full-length PRO1057 sequenceshown in FIG. 118 (SEQ ID NO:296) evidences the presence of thefollowing: a signal peptide from about amino acid 1 to about amino acid16, potential N-glycosylation sites from about amino acid 90 to aboutamino acid 93, from about amino acid 110 to about amino acid 113 andfrom about amino acid 193 to about amino acid 196, a glycosaminoglycanattachment site from about amino acid 236 to about amino acid 239 and aserine protease histidine-containing active site from about amino acid165 to about amino acid 170. Clone UNQ522 (DNA57253-1382) has beendeposited with ATCC on May 14, 1998 and is assigned ATCC deposit no.209867.

Analysis of the amino acid sequence of the full-length PRO1057polypeptide suggests that it possesses significant sequence similarityto various protease proteins, thereby indicating that PRO1057 may be anovel protease. More specifically, an analysis of the Dayhoff database(version 35.45 SwissProt 35) evidenced significant homology between thePRO1057 amino acid sequence and the following Dayhoff sequences,TRYE_DROER, P_R14159, A69660, EBN1_EBV, S65494, GEN12688, A51084_(—)1,P_R99571, A57514 and AF003200_(—)1.

Example 47 Isolation of cDNA Clones Encoding Human PRO1071

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA53035. Based on the DNA53035 consensus sequence,it was determined that that consensus sequence shared significantsequence identity with Incyte EST clone no. 2872569, a clone that uponreview appeared to encode a full length protein. As such, Incyte ESTclone no. 2872569 was purchased and its insert was obtained andsequenced so as to confirm the proper sequence. This sequence is hereindesignated UNQ528 or DNA58847-1383.

DNA sequencing of the clone isolated as described above gave thefull-length DNA sequence for PRO1071 [herein designated as UNQ528(DNA58847-1383)] (SEQ ID NO:300) and the derived protein sequence forPRO1071.

The entire nucleotide sequence of UNQ528 (DNA58847-1383) is shown inFIG. 119 (SEQ ID NO:300). Clone UNQ528 (DNA58848-1383) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 133–135 and ending at the stop codon at nucleotidepositions 1708–1710 (FIG. 119). The predicted polypeptide precursor is525 amino acids long (FIG. 120). The full-length PRO1071 protein shownin FIG. 120 has an estimated molecular weight of about 58,416 daltonsand a pI of about 6.62. Analysis of the full-length PRO1071 sequenceshown in FIG. 120 (SEQ ID NO:301) evidences the presence of thefollowing: a signal peptide from about amino acid 1 to about amino acid25, a potential N-glycosylation site from about amino acid 251 to aboutamino acid 254, a thrombospondin-1 homology block from about amino acid385 to about amino acid 399 and von Willibrands factor type C homologyblocks from about amino acid 385 to about amino acid 399, from aboutamino acid 445 to about amino acid 459 and from about amino acid 42 toabout amino acid 56. Clone UNQ528 (DNA58847-1383) has been depositedwith ATCC on May 20, 1998 and is assigned ATCC deposit no. 209879.

Analysis of the amino acid sequence of the full-length PRO1071polypeptide suggests that it possesses significant sequence similarityto the thrombospondin protein, thereby indicating that PRO1071 may be anovel thrombospondin homolog. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO1071 amino acid sequence and the followingDayhoff sequences, AB002364_(—)1, D67076_(—)1, BTPCINPGN_(—)1,CET13H10_(—)1, CEF25H8_(—)5, CEF53B6_(—)2, CEC26C6_(—)6, HSSEMG_(—)1,CET21B6_(—)4 and BTY08561_(—)1.

Example 48 Isolation of cDNA Clones Encoding Human PRO1072

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA53125. Based on the DNA53125 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1072.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CCAGGAAATGCTCCAGGAAGAGCC-3′ (SEQ ID NO:305)-   reverse PCR primer 5′-GCCCATGACACCAAATTGAAGAGTGG-3′ (SEQ ID NO:306)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA53125 sequence which had the    following nucleotide sequence    hybridization probe-   5′-AACGCAGGGATCTTCCAGTGCCCTTACATGAAGACTGAAGATGGG-3′ (SEQ ID NO:307)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1072 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue (LIB26).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1072 [herein designated as UNQ529(DNA58747-1384)] (SEQ ID NO:302) and the derived protein sequence forPRO1072.

The entire nucleotide sequence of UNQ529 (DNA58747-1384) is shown inFIG. 121 (SEQ ID NO:302). Clone UNQ529 (DNA58747-1384) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 65–67 and ending at the stop codon at nucleotidepositions 1073–1075 (FIG. 121). The predicted polypeptide precursor is336 amino acids long (FIG. 122). The full-length PRO1072 protein shownin FIG. 122 has an estimated molecular weight of about 36,865 daltonsand a pI of about 9.15. Analysis of the full-length PRO1072 sequenceshown in FIG. 122 (SEQ ID NO:303) evidences the presence of thefollowing: a signal peptide from about amino acid 1 to about amino acid21, short-chain alcohol dehydrogenase protein homology blocks from aboutamino acid 134 to about amino acid 144, from about amino acid 44 toabout amino acid 56 and from about amino acid 239 to about amino acid248 and potential N-glycosylation sites from about amino acid 212 toabout amino acid 215 and from about amino acid 239 to about amino acid242. Clone UNQ529 (DNA58747-1384) has been deposited with ATCC on May14, 1998 and is assigned ATCC deposit no. 209868.

Analysis of the amino acid sequence of the full-length PRO1072polypeptide suggests that it possesses significant sequence similarityto the reductase family of proteins, thereby indicating that PRO1072 maybe a novel reductase. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO1072 amino acid sequence and the following Dayhoffsequences, P_W03198, P_W15759, P_R60800, MTV037_(—)3, CEC15H11_(—)6,ATAC00234314, MTV022_(—)13, SCU43704_(—)1, OXIR_STRAT AND CELC01G8_(—)3.

Example 49 Isolation of cDNA Clones Encoding Human PRO1075

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA34363. Based on the DNA34363 sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1075.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-TGAGAGGCCTCTCTGGAAGTTG-3′ (SEQ ID NO:312)-   forward PCR primer 5′-GTCAGCGATCAGTGAAAGC-3′ (SEQ ID NO:313)-   forward PCR primer 5′-CCAGAATGAAGTAGCTCGGC-3′ (SEQ ID NO:314)-   forward PCR primer 5′-CCGACTCAAAATGCATTGTC-3′ (SEQ ID NO:315)-   forward PCR primer 5′-CATTTGGCAGGAATTGTCC-3′ (SEQ ID NO:316)-   forward PCR primer 5′-GGTGCTATAGGCCAAGGG-3′ (SEQ ID NO:317)-   reverse PCR Primer 5′-CTGTATCTCTGGGCTATGTCAGAG-3′ (SEQ ID NO:318)-   reverse PCR primer 5′-CTACATATAATGGCACATGTCAGCC-3′ (SEQ ID NO:319)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA34363 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CGTCTTCCTATCCTTACCCGACCTCAGATGCTCCCTTCTGCTCCTG-3′ (SEQ ID NO:320)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1075 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human skin tumor tissue (LIB324).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1075 [herein designated as UNQ532(DNA57689-1385)] (SEQ ID NO:308) and the derived protein sequence forPRO1075.

The entire nucleotide sequence of UNQ532 (DNA57689-1385) is shown inFIG. 124 (SEQ ID NO:308). Clone UNQ532 (DNA57689-1385) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 137–139 and ending at the stop codon at nucleotidepositions 1355–1357 (FIG. 124). The predicted polypeptide precursor is406 amino acids long (FIG. 125). The full-length PRO1075 protein shownin FIG. 125 has an estimated molecular weight of about 46,927 daltonsand a pI of about 5.21. Analysis of the full-length PRO1075 sequenceshown in FIG. 125 (SEQ ID NO:309) evidences the presence of thefollowing: a signal peptide from about amino acid 1 to about amino acid29, an endoplasmic reticulum targeting sequence from about amino acid403 to about amino acid 406, a tyrosine kinase phosphorylation site fromabout amino acid 203 to about amino acid 211 and a sequence block havinghomology to the thioredoxin family of proteins from about amino acid 50to about amino acid 66. Clone UNQ532 (DNA57689-1385) has been depositedwith ATCC on May 14, 1998 and is assigned ATCC deposit no. 209869.

Analysis of the amino acid sequence of the full-length PRO1075polypeptide suggests that it possesses significant sequence similarityto protein disulfide isomerase, thereby indicating that PRO1075 may be anovel protein disulfide isomerase. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO1075 amino acid sequence and the followingDayhoff sequences, CELC30H7_(—)2, CELC06A6_(—)3, CELF42G8_(—)3, S57942,ER72_CAEEL, CELC07A12_(—)3, CEH06O₀₁ _(—)4 and P_R51696.

Example 50 Isolation of cDNA Clones Encoding Human PRO181

A cDNA sequence isolated in the amylase screen described in Example 2above was found, by BLAST and FastA sequence alignment, to have sequencehomology to a nucleotide sequence encoding the cornichon protein. ThiscDNA sequence is herein designated DNA13242 (FIG. 130; SEQ ID NO:323).Based on the sequence homology, oligonucleotide probes were generatedfrom the sequence of the DNA13242 molecule and used to screen a humanplacenta (LIB89) library prepared as described in paragraph 1 of Example2 above. The cloning vector was pRK5B (pRK5B is a precursor of pRK5Dthat does not contain the SfiI site; see, Holmes et al., Science,253:1278–1280 (1991)), and the cDNA size cut was less than 2800 bp.

The oligonucleotide probes employed included:

-   forward PCR primer 5′-GTGCAGCAGAGTGGCTTACA-3′ (SEQ ID NO:326)-   reverse PCR primer 5′-ACTGGACCAATTCTTCTGTG-3′ (SEQ ID NO:327)    hybridization probe-   5′-GATATTCTAGCATATTGTCAGAAGGAAGGATGGTGCAAATTAGCT-3′ (SEQ ID NO:328)

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 14–16 and ending at the stop codon found at nucleotidepositions 446–448 (FIG. 128; SEQ ID NO:321). The predicted polypeptideprecursor is 144 amino acids long, has a calculated molecular weight ofapproximately 16,699 daltons and an estimated pI of approximately 5.6.Analysis of the full-length PRO181 sequence shown in FIG. 129 (SEQ IDNO:322) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 20, a putative type IItransmembrane domain from about amino acid 11 to about amino acid 31 andother transmembrane domains from about amino acid 57 to about amino acid77 and from about amino acid 123 to about amino acid 143. Clone UNQ155(DNA23330-1390) has been deposited with ATCC on Apr. 14, 1998 and isassigned ATCC deposit no. 209775.

Analysis of the amino acid sequence of the full-length PRO181polypeptide suggests that it possesses significant sequence similarityto the cornichon protein, thereby indicating that PRO181 may be a novelcornichon homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO181 amino acid sequence and the following Dayhoffsequences, AF022811_(—)1, CET09E8_(—)3, S64058, YGF4_YEAST, YB60_YEAST,EBU89455_(—)1, SIU36383_(—)3 and PH1371.

Example 51 Isolation of cDNA Clones Encoding Human PRO195

A cDNA sequence was isolated in the amylase screen described in Example2 above and is herein designated DNA13199 (FIG. 134; SEQ ID NO:332). TheDNA13199 sequence was then compared to a variety of expressed sequencetag (EST) databases which included public EST databases (e.g., GenBank)to identify existing homologies. The homology search was performed usingthe computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460–480 (1996)). Those comparisons resulting in a BLASTscore of 70 (or in some cases 90) or greater that did not encode knownproteins were clustered and assembled into consensus DNA sequences withthe program “phrap” (Phil Green, University of Washington, Seattle,Wash.). The consensus sequence obtained therefrom is herein designatedas DNA22778.

Based on the DNA22778 sequence, oligonucleotide probes were generatedand used to screen a human placenta library (LIB89) prepared asdescribed in paragraph 1 of Example 2 above. The cloning vector waspRK5B (pRK5B is a precursor of pRK5D that does not contain the SfiIsite; see, Holmes et al., Science, 253:1278–1280 (1991)), and the cDNAsize cut was less than 2800 bp.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-ACAAGCTGAGCTGCTGTGACAG-3′ (SEQ ID NO:333)-   reverse PCR primer 5′-TGATTCTGGCAACCAAGATGGC-3′ (SEQ ID NO:334)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA22778 sequence which had the following    nucleotide sequence    hybridization probe-   5′-ATGGCCTrGGCCGGAGGTTCGGGGACCGCTTCGGCTGAAG-3′ (SEQ ID NO:335)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO195 gene using the probe oligonucleotideand one of the PCR primers.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 70–72 and ending at the stop codon found at nucleotidepositions 1039–1041 (FIG. 132; SEQ ID NO:330). The predicted polypeptideprecursor is 323 amino acids long, has a calculated molecular weight ofapproximately 36,223 daltons and an estimated pI of approximately 5.06.Analysis of the full-length PRO195 sequence shown in FIG. 132 (SEQ IDNO:330) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 31, a transmembrane domain fromabout amino acid 241 to about amino acid 260 and a potentialN-glycosylation site from about amino acid 90 to about amino acid 93.Clone UNQ169 (DNA26847-1395) has been deposited with ATCC on Apr. 14,1998 and is assigned ATCC deposit no. 209772.

Analysis of the amino acid sequence of the full-length PRO195polypeptide suggests that it possesses no significant sequencesimilarity to any known protein. However, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced some degree of homologybetween the PRO195 amino acid sequence and the following Dayhoffsequences, P_P91380, AF035118_(—)1, HUMTROPCS_(—)1, NUOD_SALTY andE70002.

Example 52 Isolation of cDNA Clones Encoding Human PRO865

A cDNA sequence isolated in the amylase screen described in Example 2above was herein designated DNA37642 (FIG. 137, SEQ ID NO:338). TheDNA37642 sequence was then compared to a variety of expressed sequencetag (EST) databases which included public EST databases (e.g., GenBank)and a proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals,Palo Alto, Calif.) to identify homologies therebetween. The homologysearch was performed using the computer program BLAST or BLAST2 (Altshulet al., Methods in Enzymology 266:460–480 (1996)). Those comparisonsresulting in a BLAST score of 70 (or in some cases 90) or greater thatdid not encode known proteins were clustered and assembled intoconsensus DNA sequences with the program “phrap” (Phil Green, Universityof Washington, Seattle, Wash.). The consensus sequence obtained isherein designated DNA48615.

Based on the DNA48615 consensus sequence, probes were generated and usedto screen a human fetal kidney (LIB227) library prepared as described inparagraph 1 of Example 2 above. The cloning vector was pRK5B (pRK5B is aprecursor of pRK5D that does not contain the SfiI site; see, Holmes etal., Science, 253:1278–1280 (1991)), and the cDNA size cut was less than2800 bp.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 1 5′-AAGCTGCCGGAGCTGCAATG-3′ (SEQ ID NO:339)-   forward PCR primer 2 5′-TTGCTTCTTAATCCTGAGCGC-3′ (SEQ ID NO:340)-   forward PCR primer 3 5′-AAAGGAGGACTTTCGACTGC-3′ (SEQ ID NO:341)-   reverse PCR primer 1 5′-AGAGATTCATCCACTGCTCCAAGTCG-3′ (SEQ ID    NO:342)-   reverse PCR primer 2 5′-TGTCCAGAAACAGGCACATATCAGC-3′ (SEQ ID NO:343)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA48615 sequence which had the    following nucleotide sequence    hybridization probe-   5′-AGACAGCGGCACAGAGGTGCTTCTGCCAGGTTAGTGGTTACTTGGATGAT-3′ (SEQ ID    NO:344)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO865 gene using the probe oligonucleotideand one of the PCR primers.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 173–175 and ending at the stop codon found at nucleotidepositions 1577–1579 (FIG. 135; SEQ ID NO:336). The predicted polypeptideprecursor is 468 amino acids long, has a calculated molecular weight ofapproximately 54,393 daltons and an estimated pI of approximately 5.63.Analysis of the full-length PRO865 sequence shown in FIG. 136 (SEQ IDNO:337) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 23, potential N-glycosylationsites from about amino acid 280 to about amino acid 283 and from aboutamino acid 384 to about amino acid 387, a potential amidation site fromabout amino acid 94 to about amino acid 97, glycosaminoglycan attachmentsites from about amino acid 20 to about amino acid 23 and from aboutamino acid 223 to about amino acid 226, an aminotransferase class-Vpyridoxyl-phosphate amino acid sequence block from about amino acid 216to about amino acid 222 and an amino acid sequence block similar to thatfound in the interleukin-7 protein from about amino acid 338 to aboutamino acid 343. Clone UNQ434 (DNA53974-1401) has been deposited withATCC on Apr. 14, 1998 and is assigned ATCC deposit no. 209774.

Analysis of the amino acid sequence of the full-length PRO865polypeptide suggests that it possesses no significant sequencesimilarity to any known protein. However, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced some degree of homologybetween the PRO865 amino acid sequence and the following Dayhoffsequences, YMN0_YEAST, ATFCA4_(—)43, S44168, P_W14549 and RABTCRG4_(—)1.

Example 53 Isolation of cDNA Clones Encoding Human PRO827

A cDNA sequence isolated in the amylase screen described in Example 2above was found, by BLAST and FastA sequence alignment, to have sequencehomology to nucleotide sequences encoding various integrin proteins.This cDNA sequence is herein designated DNA47751 (see FIG. 140; SEQ IDNO:347). Based on the sequence homology, probes were generated from thesequence of the DNA47751 molecule and used to screen a human fetalpigment epithelium library (LIB113) prepared as described in paragraph 1of Example 2 above. The cloning vector was pRK5B (pRK5B is a precursorof pRK5D that does not contain the SfiI site; see, Holmes et al.,Science 253:1278–1280 (1991)), and the cDNA size cut was less than 2800bp.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-AGGGACAGAGGCCAGAGGACTTC-3′ (SEQ ID NO:348)-   reverse PCR primer 5′-CAGGTGCATATTCACAGCAGGATG-3′ (SEQ ID NO:349)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA47751 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GGAACTCCCCTTCGTCACTCACCTGTTCTTGCCCCTGGTGTTCCT-3′ (SEQ ID NO:350)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO827 gene using the probe oligonucleotideand one of the PCR primers.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 134–136 and ending at the stop codon found at nucleotidepositions 506–508 (FIG. 138; SEQ ID NO:345). The predicted polypeptideprecursor is 124 amino acids long, has a calculated molecular weight ofapproximately 13,352 daltons and an estimated pI of approximately 5.99.Analysis of the full-length PRO827 sequence shown in FIG. 139 (SEQ IDNO:346) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 22, a cell attachment sequencefrom about amino acid 70 to about amino acid 72, a potentialN-glycosylation site from about amino acid 98 to about amino acid 101and an integrin alpha chain protein homology sequence from about aminoacid 67 to about amino acid 81. Clone UNQ468 (DNA57039-1402) has beendeposited with ATCC on Apr. 14, 1998 and is assigned ATCC deposit no.209777.

Analysis of the amino acid sequence of the full-length PRO827polypeptide suggests that it possesses significant sequence similarityto the VLA-2 integrin protein and various other integrin proteins,thereby indicating that PRO827 may be a novel integrin or splice variantthereof. More specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35) evidenced significant homology between the PRO240amino acid sequence and the following Dayhoff sequences, S44142,ITA2_HUMAN, ITA1_RAT, ITA1_HUMAN, ITA4_HUMAN, ITA9_HUMAN, AF032108_(—)1,ITAM_MOUSE, ITA8_CHICK and ITA6_CHICK.

Example 54 Isolation of cDNA Clones Encoding Human PRO1114

A cDNA sequence isolated in the amylase screen described in Example 2was found, by the WU-BLAST2 sequence alignment computer program, to havecertain sequence identity to other known interferon receptors. This cDNAsequence is herein designated DNA48466 (FIG. 143; SEQ ID NO:352). Basedon the sequence identity, probes were generated from the sequence of theDNA48466 molecule and used to screen a human breast carconoma library(LIB135) prepared as described in paragraph 1 of Example 2 above. Thecloning vector was pRK5B (pRK5B is a precursor of pRK5D that does notcontain the SfiI site; see, Holmes et al., Science, 253:1278–1280(1991)), and the cDNA size cut was less than 2800 bp.

The oligonucleotide probes employed were as follows:

-   forward PCR primer 5′-AGGCTTCGCTGCGACTAGACCTC-3′ (SEQ ID NO:354)-   reverse PCR primer 5′-CCAGGTCGGGTAAGGATGGTTGAG-3′ (SEQ ID NO:355)    hybridization probe-   5′-TTTCTACGCATTGATTCCATGTTTGCTCACAGATGAAGTGGCCATTCTGC-3′ (SEQ ID    NO:356)

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 250–252, and a stop signal at nucleotide positions 1183–1185(FIG. 141, SEQ ID NO:351). The predicted polypeptide precursor is 311amino acids long, has a calculated molecular weight of approximately35,076 daltons and an estimated pI of approximately 5.04. Analysis ofthe full-length PRO1114 interferon receptor sequence shown in FIG. 142(SEQ ID NO:352) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 29, a transmembranedomain from about amino acid 230 to about amino acid 255, potentialN-glycosylation sites from about amino acid 40 to about amino acid 43and from about amino acid 134 to about amino acid 137, an amino acidsequence block having homology to tissue factor proteins from aboutamino acid 92 to about amino acid 119 and an amino acid sequence blockhaving homology to integrin alpha chain proteins from about amino acid232 to about amino acid 262. Clone UNQ557 (DNA57033–1403) has beendeposited with ATCC on May 27, 1998 and is assigned ATCC deposit no.209905.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 142 (SEQ ID NO:352), evidenced significant homologybetween the PRO1114 interferon receptor amino acid sequence and thefollowing Dayhoff sequences: G01418, INR1_MOUSE, P_R71035, INGS_HUMAN,A26595_(—)1, A26593_(—)1, I56215 and TF_HUMAN.

Example 55 Isolation of cDNA Clones Encoding Human PRO237

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA30905. Based on the DNA30905 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO237.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-TCTGCTGAGGTGCAGCTCATTCAC-3′ (SEQ ID NO:359)-   reverse PCR primer 5′-GAGGCTCTGGAAGATCTGAGATGG-3′ (SEQ ID NO:360)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA30905 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GCCTCTTTGTCAACGTTGCCAGTACCTCTAACCCATTCCrCAGTCGCCTC-3′ (SEQ ID    NO:361)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO237 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal brain tissue (L[B153).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO237 [herein designated as UNQ211(DNA34353-1428)] (SEQ ID NO:357) and the derived protein sequence forPRO237.

The entire nucleotide sequence of UNQ211 (DNA34353-1428) is shown inFIG. 144 (SEQ ID NO:357). Clone UNQ211 (DNA34353-1428) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 586–588 and ending at the stop codon at nucleotidepositions 1570–1572 (FIG. 144). The predicted polypeptide precursor is328 amino acids long (FIG. 145). The full-length PRO237 protein shown inFIG. 145 has an estimated molecular weight of about 36,238 daltons and apI of about 9.90. Analysis of the full-length PRO237 sequence shown inFIG. 145 (SEQ ID NO:358) evidences the presence of the following: asignal peptide from about amino acid 1 to about amino acid 23, atransmembrane domain from about amino acid 177 to about amino acid 199,potential N-glycosylation sites from about amino acid 118 to about aminoacid 121, from about amino acid 170 to about amino acid 173 and fromabout amino acid 260 to about amino acid 263 and eukaryotic-typecarbonic anhydrase sequence homology blocks from about amino acid 222 toabout amino acid 270, from about amino acid 128 to about amino acid 164and from about amino acid 45 to about amino acid 92. Clone UNQ211(DNA34353-1428) has been deposited with ATCC on May 12, 1998 and isassigned ATCC deposit no. 209855.

Analysis of the amino acid sequence of the full-length PRO237polypeptide suggests that it possesses significant sequence similarityto the carbonic anhydrase protein. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO237 amino acid sequence and the followingDayhoff sequences, AF050106_(—)1, OACALP_(—)1, CELD1022_(—)8,CAH2_HUMAN, 1CAC, CAH5_HUMAN, CAHP_HUMAN, CAH3_HUMAN, CAH1_HUMAN and2CAB.

Example 56 Isolation of cDNA Clones Encoding Human PRO541

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42259. Based on the DNA42259 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO541.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GGACAGAATTTGGGAGCACACTGG-3′ (SEQ ID NO:364)-   forward PCR primer 5′-CCAAGAGTATACTGTCCTCG-3′ (SEQ ID NO:365)-   reverse PCR primer 5′-AGCACAGATTTTCTCTACAGCCCCC-3′ (SEQ ID NO:366)-   reverse PCR primer 5′-AACCACTCCAGCATGTACTGCTGC-3′ (SEQ ID NO:367)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA42259 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CCATTCAGGTGTTCTGGCCCTGTATGTACACATTATACACAGGTCGTGTG-3′ (SEQ ID    NO:368)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO541 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO541 [herein designated as UNQ342(DNA45417-1432)] (SEQ ID NO:362) and the derived protein sequence forPRO541.

The entire nucleotide sequence of UNQ342 (DNA45417-1432) is shown inFIG. 146 (SEQ ID NO:362). Clone UNQ342 (DNA45417-1432) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 469–471 and ending at the stop codon at nucleotidepositions 1969–1971 (FIG. 146). The predicted polypeptide precursor is500 amino acids long (FIG. 147). The full-length PRO541 protein shown inFIG. 147 has an estimated molecular weight of about 56,888 daltons and apI of about 8.53. Analysis of the full-length PRO541 sequence shown inFIG. 147 (SEQ ID NO:363) evidences the presence of the following: asignal peptide from about amino acid 1 to about amino acid 20, aminoacid sequence blocks having homology to extracellular proteinsSCP/Tpx-1/Ag5/PR-1/Sc7 from about amino acid 165 to about amino acid186, from about amino acid 196 to about amino acid 218, from about aminoacid 134 to about amino acid 146, from about amino acid 96 to aboutamino acid 108 and from about amino acid 58 to about amino acid 77 and apotential N-glycosylation site from about amino acid 28 to about aminoacid 31. Clone UNQ342 (DNA45417-1432) has been deposited with ATCC onMay 27, 1998 and is assigned ATCC deposit no. 209910.

Analysis of the amino acid sequence of the full-length PRO541polypeptide suggests that it possesses significant sequence similarityto a trypsin inhibitor protein, thereby indicating that PRO541 may be anovel trypsin inhibitor. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO541 amino acid sequence and the following Dayhoffsequences, D45027_(—)1, AB009609_(—)1, JC5308, CRS3_HORSE, TPX1_HUMAN,HELO_HELHO, GEN14351, A28112_(—)1, CET05A10_(—)4 and P_W11485.

Example 57 Isolation of cDNA Clones Encoding Human PRO273

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA36465. Based on the DNA36465 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO273.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CAGCGCCCTCCCCATGTCCCTG-3′ (SEQ ID NO:371)-   reverse PCR primer 5′-TCCCAACTGGTTTGGAGTTTCCC-3′ (SEQ ID NO:372)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA36465 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CTCCGGTCAGCATGAGGCTCCTGGCGGCCGCTGCTCCTGCTGCTG-3′ (SEQ ID NO:373)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO273 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO273 [herein designated as UNQ240(DNA39523-1192)] (SEQ ID NO:369) and the derived protein sequence forPRO273.

The entire nucleotide sequence of UNQ240 (DNA39523-1192) is shown inFIG. 148 (SEQ ID NO:369). Clone UNQ240 (DNA39523-1192) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 167–169 and ending at the stop codon at nucleotidepositions 500–502 (FIG. 148). The predicted polypeptide precursor is 111amino acids long (FIG. 149). Clone UNQ240 (DNA39523-1192) has beendeposited with the ATCC. It is understood that the deposited clonecontains the actual sequence and that the sequences provided herein aremerely representative based on current sequencing techniques. Moreover,given the sequences provided herein and knowledge of the universalgenetic code, the corresponding nucleotides for any given amino acid canbe routinely identified and vice versa.

Analysis of the amino acid sequence of the full-length PRO273polypeptide suggests that portions of it possess sequence identity withhuman macrophage inflammatory protein-2, cytokine-induced neutrophilchemoattractant 2, and neutrophil chemotactic factor 2-beta, therebyindicating that PRO273 is a novel chemokine.

As discussed further below, the cDNA was subcloned into a baculovirusvector and expressed in insect cells as a C-terminally tagged IgG fusionprotein. N-terminal sequencing of the resultant protein identified thesignal sequence cleavage site, yielding a mature polypeptide of 77 aminoacids. The mature sequence, showing 31–40% identity to other human CXCchemokines, includes the four canonical cysteine residues but lacks theELR motif. Northern analysis demonstrates expression at least in thesmall intestine, colon, spleen, lymph node and kidney. By in situhybridization, also described in detail below, mRNA is localized to thelamina propria of intestinal villi and to renal tubules.

Example 58 Isolation of cDNA Clones Encoding Human PRO701

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA39848. Based on the DNA39848 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO701.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GGCAAGCTACGGAAACGTCATCGTG-3′ (SEQ ID NO:376)-   reverse PCR primer 5′-AACCCCCGAGCCAAAAGATGGTCAC-3′ (SEQ ID NO:377)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA39848 sequence which had the    following nucleotide sequence:    hybridization probe-   5′-GTACCGGTGACCAGGCAGCAAAAGGCAACTATGGGCTCCTGGATCAG-3′ (SEQ ID    NO:378).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO701 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO701 [herein designated as UNQ365(DNA44205-1285)] (SEQ ID NO:374) and the derived protein sequence forPRO701.

The entire nucleotide sequence of UNQ365 (DNA44205-1285) is shown inFIG. 150 (SEQ ID NO:374). Clone UNQ365 (DNA44205-1285) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 50–52 and ending at the stop codon at nucleotidepositions 2498–3000 (FIG. 150). The predicted polypeptide precursor is816 amino acids long (FIG. 151). The full-length PRO701 protein shown inFIG. 151 has an estimated molecular weight of about 91,794 daltons, a pIof about 5.88 and NX(S/T) being 4. Clone UNQ365 (DNA44205-1285) has beendeposited with the ATCC on Mar. 31, 1998. It is understood that theclone was the correct and actual sequence, wherein the sequencesprovided herein are representative based on sequencing techniques.

Still regarding the amino acid sequence shown in FIG. 151, there is apotential signal peptide cleavage site at about amino acid 25. There arepotential N-glycosylation sites at about amino acid positions 83, 511,716 and 803. The carboxylesterases type-B signature 2 sequence is atabout residues 125 to 135. Regions homologous with carboxylesterasetype-B are also at about residues 54–74, 197–212 and 221–261. Apotential transmembrane region corresponds approximately to amino acids671 through about 700. The corresponding nucleic acids can be routinelydetermined from the sequences provided herein.

Analysis of the amino acid sequence of the full-length PRO701polypeptide suggests that it possess significant homology to theneuroligins from rattus norvegicus indicating that PRO701 may be a novelhuman neuroligin.

Example 59 Isolation of cDNA Clones Encoding Human PRO704

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43033. Based on the DNA43033 consensus sequence,oligonucleotides were synthesized: 1) to identity by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO704.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CCTTGGGTCGTGGCAGCAGTGG-3′ (SEQ ID NO:381);-   reverse PCR primer 5′-CACTCTCCAGGCTGCATGCTCAGG-3′ (SEQ ID NO:382).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA43033 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-GTCAAACGTTCGAGTACTTGAAACGGGAGCACTCGCTGTCGAAGC-3′ (SEQ ID NO:383).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO704 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO704 [herein designated as UNQ368(DNA50911-1288)] (SEQ ID NO:379) and the derived protein sequence forPRO704.

The entire nucleotide sequence of UNQ368 (DNA50911-1288) is shown inFIG. 152 (SEQ ID NO:379). Clone UNQ368 (DNA50911-1288) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 8–10 and ending at the stop codon at nucleotidepositions 1052–1054 (FIG. 152). The predicted polypeptide precursor is348 amino acids long (FIG. 153). The full-length PRO704 protein shown inFIG. 153 has an estimated molecular weight of about 39,711 and a pI ofabout 8.7. Clone UNQ368 (DNA50911-1288) has been deposited with the ATCCon Mar. 31, 1998. Regarding the sequence, it is understood that thedeposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO704polypeptide suggests that portions of it possess significant homology tothe vesicular integral membrane protein 36, thereby indicating thatPRO704 may be a novel vesicular integral membrane protein.

Still analyzing the amino acid sequence of SEQ ID NO:380, the putativesignal peptide is at about amino acids 1–39 of SEQ ID NO:380. Thetransmembrane domain is at amino acids 310–335 of SEQ ID NO:380. Apotential N-glycosylation site is at about amino acids 180–183 of SEQ IDNO:380. The corresponding nucleotides can be routinely determined giventhe sequences provided herein.

Example 60 Isolation of cDNA Clones Encoding Human PRO706

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA40669. Based on the DNA40669 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO706.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CCAAGCAGCTTAGAGCTCCAGACC-3′ (SEQ ID NO:386)-   reverse PCR primer 5′-TTCCCTATGCTCTGTATTGGCATGG-3′ (SEQ ID NO:387)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA40669 sequence which had the    following nucleotide sequence    hybridization probe-   5′-GCCACTTCTGCCACAATGTCAGCTTTCCCTGTACCAGAAATGGCTGTGTT-3′ (SEQ ID    NO:388)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO706 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal brain tissue (LIB153).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO706 therein designated as UNQ370(DNA48329-1290)] (SEQ ID NO:384) and the derived protein sequence forPRO706. It is understood that the deposited clone contains the actualsequence, and that the sequences provided herein are representativebased on current sequencing techniques.

The entire nucleotide sequence of UNQ370 (DNA48329-1290) is shown inFIG. 154 (SEQ ID NO:384). Clone UNQ370 (DNA48329-1290) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 279–281 and ending at the stop codon at nucleotidepositions 1719–1721 (FIG. 154). The predicted polypeptide precursor is480 amino acids long (FIG. 155). The full-length PRO706 protein shown inFIG. 155 has an estimated molecular weight of about 55,239 daltons and apI of about 9.30. Clone UNQ370 (DNA48329-1290) has been deposited withthe ATCC on Apr. 21, 1998.

Still regarding the amino acid sequence shown in FIG. 155, there is apotential signal peptide cleavage site at about amino acid 19. There arepotential N-glycosylation sites at about amino acid positions 305 and354. There is a potential tyrosine kinase phosphorylation site at aboutamino acid position 333. A region homologous with histidine acidphosphatase is at about residues 87–102. The corresponding nucleic acidregions can be routinely determined given the provided sequences, i.e.,the codons can be determined from the specifically named amino acidsgiven.

Analysis of the amino acid sequence of the full-length PRO706polypeptide suggests that portions of it possess significant homology tothe human prostatic acid phosphatase precursor thereby indicating thatPRO706 may be a novel human prostatic acid phosphatase.

Example 61 Isolation of cDNA Clones Encoding Human PRO707

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42775. Based on DNA42775, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO707.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-TCCGTCTCTGTGAACCGCCCCAC-3′ (SEQ ID NO:391);-   reverse PCR primer 5′-CTCGGGCGCATTGTCGTTCTGGTC-3′ (SEQ ID NO:392).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA42775 sequence which had the following    nucleotide sequence:    hybridization probe-   5′-CCGACTGTGAAAGAGAACGCCCCAGATCCACTTATTCCCC-3′ (SEQ ID NO:393).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO707 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO707 [herein designated as UNQ371(DNA48306-1291)] (SEQ ID NO:389) and the derived protein sequence forPRO707.

The entire nucleotide sequence of UNQ371 (DNA48306-1291) is shown inFIG. 156 (SEQ ID NO:389). Clone UNQ371 (DNA48306-1291) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 371–373 and ending at the stop codon at nucleotidepositions 3119–3121 of SEQ ID NO:389. The predicted polypeptideprecursor is 916 amino acids long (FIG. 157). The full-length PRO707protein shown in FIG. 157 has an estimated molecular weight of about100,204 daltons and a pI of about 4.92. Clone UNQ371 (DNA48306-1291) hasbeen deposited with ATCC on May 27, 1998. It is understood that theclone UNQ371 which is deposited is that which encodes PRO707, and thatthe sequences herein are merely representations based on knownsequencing techniques which may be subject to minor errors.

Regarding analysis of the amino acid sequence, the signal sequenceappears to be at about 1 through 30 of SEQ ID NO:390. Cadherinsextracellular repeated domain signature sequence is at about amino acids121–131, 230–240, 335–345, 440–450, and 550–560 of SEQ ID NO:390.Tyrosine kinase phosphorylation sites are at about amino acids 124–132and 580–586 of SEQ ID NO:390. A potential transmembrane domain is atabout amino acids 682–715±5. The nucleic acid positions can be derivedby referring to the corresponding codon for the named amino acid.

Analysis of the amino acid sequence of the full-length PRO707polypeptide suggests that portions of it possess significant homology tothe cadherin FIB3 protein, expressed in human fibroblasts, therebyindicating that PRO707 may be a novel cadherin.

Example 62 Isolation of cDNA Clones Encoding Human PRO322

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA48336. Based on the DNA48336 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO322.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CAGCCTACAGAATAAAGATGGCCC-3′ (SEQ ID NO:396)-   reverse PCR primer 5′-GGTGCAATGATCTGCCAGGCTGAT-3′ (SEQ ID NO:397)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA48336 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-AGAAATACCTGTGGTTCAGTCCATCCCAAACCCCTGCTACAACAGCAG-3′ (SEQ ID    NO:398).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO322 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO322 [herein designated as UNQ283(DNA48336-1309)] (SEQ ID NO:394) and the derived protein sequence forPRO322. It is understood that UNQ283 (DNA48336-1309) in fact encodesPRO322, and that SEQ ID NO:394 is a representation of the sequence basedon sequencing techniques known in the art.

The entire nucleotide sequence of UNQ283 (DNA48336-1309) is shown inFIG. 158 (SEQ ID NO:394). Clone UNQ283 (DNA48336-1309) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 166–168 and ending at the stop codon at nucleotidepositions 946–948 (FIG. 158). The predicted polypeptide precursor is 260amino acids long (FIG. 159). The full-length PRO322 protein shown inFIG. 159 has an estimated molecular weight of about 28,028 daltons and apI of about 7.87. Clone UNQ283 (DNA48336-1309) has been deposited withATCC and is assigned ATCC deposit no. 209669.

Regarding the amino acid sequence of FIG. 159, a potentialN-glycosylation site is at amino acid 110 of SEQ ID NO:395. The serineproteases, trypsin family and histidine active site is identified atamino acids 69 through 74 of SEQ ID NO:395 and the consensus sequence isidentified at amino acids 207 through 217 of SEQ ID NO:395. The kringledomain proteins motif is identified at amino acids 205 through 217 ofSEQ ID NO:395. The putative signal peptide is encoded at about aminoacids 1–23.

Analysis of the amino acid sequence of the full-length PRO322polypeptide suggests that portions of it possess significant homology toneuropsin and other serine proteases, thereby indicating that PRO322 isa novel serine protease related to neuropsin.

Example 63 Isolation of cDNA Clones Encoding Human PRO526

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA39626. Based on the DNA39626 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO526.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-TGGCTGCCCTGCAGTACCTCTACC-3′ (SEQ ID NO:401);-   reverse PCR primer 5′-CCCTGCAGGTCATTGGCAGCTAGG-3′ (SEQ ID NO:402).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA39626 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-AGGCACTGCCTGATGACACCJTCCGCGACCTGGGCAACCTCACAC-3′ (SEQ ID NO:403).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO526 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB228).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO526 [herein designated as UNQ330(DNA44184-1319)] (SEQ ID NO:399) and the derived protein sequence forPRO526.

The entire nucleotide sequence of UNQ330 (DNA44184-1319) is shown inFIG. 160 (SEQ ID NO:399). Clone UNQ330 (DNA44184-1319) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 514–516 and ending at the stop codon at nucleotidepositions 1933–1935 (FIG. 160). The predicted polypeptide precursor is473 amino acids long (FIG. 161). The full-length PRO526 protein shown inFIG. 161 has an estimated molecular weight of about 50,708 daltons and apI of about 9.28. Clone UNQ330 (DNA44185-1319) has been deposited withthe ATCC on Mar. 26, 1998. It is understood that the clone contains theactual sequence, whereas the sequences presented herein arerepresentative based on current sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO526polypeptide suggests that portions of it possess significant homology tothe leucine repeat rich proteins including ALS, SLIT, carboxypeptidaseand platelet glycoprotein V thereby indicating that PRO526 is a novelprotein which is involved in protein-protein interactions.

Still analyzing SEQ ID NO:400, the signal peptide sequence is at aboutamino acids 1–26. A leucine zipper pattern is at about amino acids135–156. A glycosaminoglycan attachment is at about amino acids 436–439.N-glycosylation sites are at about amino acids 82–85, 179–182, 237–240and 423–426. A von Willebrand factor (VWF) type C domain(s) is found atabout amino acids 411–425. The skilled artisan can understand whichnucleotides correspond to these amino acids based on the sequencesprovided herein.

Example 64 Isolation of cDNA Clones Encoding Human PRO531

An ECD database was searched and an expressed sequence tag (EST) fromLIFESEQ™, Incyte Pharmaceuticals, Palo Alto, Calif. was identified whichshowed homology to protocadherin 3. Based on this sequence, a search wasperformed using the computer program BLAST or BLAST2 (Altshul et al.,Methods in Enzymolozy 266:460–480 (1996)) as a comparison of the ECDprotein sequences to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap. Based on the consensus sequence obtained, oligonucleotideswere synthesized: 1) to identify by PCR a cDNA library that containedthe sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PRO531.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CTGAGAACGCGCCTGAAACTGTG-3′ (SEQ ID NO:406);-   reverse PCR primer 5′-AGCGTTGTCATTGACATCGGCG-3′ (SEQ ID NO:407).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA sequence which had the following    nucleotide sequence:    hybridization probe-   5′-TTAGTTGCTCCATTCAGGAGGATCTACCCTTCCTCCTGAAATCCGCGGAA-3′ (SEQ ID    NO:408).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO531 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal brain tissue (LIB153). The cDNA librariesused to isolate the cDNA clones were constructed by standard methodsusing commercially available reagents such as those from Invitrogen, SanDiego, Calif. The cDNA was primed with oligo dT containing a NotI site,linked with blunt to Sail hemikinased adaptors, cleaved with NotI, sizedappropriately by gel electrophoresis, and cloned in a definedorientation into a suitable cloning vector (such as pRKB or pRKD; pRK5Bis a precursor of pRK5D that does not contain the SfiI site; see, Holmeset al., Science, 253:1278–1280 (1991)) in the unique XhoI and NotIsites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO531 [herein designated as UNQ332(DNA48314-1320)] (SEQ ID NO:404) and the derived protein sequence forPRO531.

The entire representative nucleotide sequence of UNQ332 (DNA48314-1320)is shown in FIG. 162 (SEQ ID NO:404). It is understood that the actualsequence is that within the clone deposited with the ATCC asDNA48314-1320. Clone UNQ332 (DNA48314-1320) contains a single openreading frame with an apparent translational initiation site atnucleotide positions 171–173 and ending at the stop codon at nucleotidepositions 2565–2567 (FIG. 162). The predicted polypeptide precursor is789 amino acids long (FIG. 163). The full-length PRO531 protein shown inFIG. 163 has an estimated molecular weight of about 87,552 daltons and apI of about 4.84. Clone UNQ332 (DNA48314-1320) has been deposited withthe ATCC on Mar. 26, 1998.

Analysis of the amino acid sequence of the full-length PRO531polypeptide suggests that portions of it possess significant homology toprotocadherin 3. Moreover, PRO531 is found in the brain, like otherprotocadherins, thereby indicating that PRO531 is a novel member of thecadherin superfamily.

Still analyzing the amino acid sequence of SEQ ID NO:405, the cadherinextracellular repeated domain signature is found at about amino acids122–132, 231–241, 336–346, 439–449 and 549–559 of SEQ ID NO:405. AnATP/GTP-binding site motif A (P-loop) is found at about amino acids285–292 of SEQ ID NO:405. N-glycosylation sites are found at least atabout amino acids 567–570, 786–790, 418–421 and 336–339 of SEQ IDNO:405. The signal peptide is at about amino acids 1–26, and thetransmembrane domain is at about amino acids 685–712 of SEQ ID NO:405.

Example 65 Isolation of cDNA Clones Encoding Human PRO534

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43038. Based on the 43048 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO534.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CACAGAGCCAGAAGTGGCGGAATC-3′ (SEQ ID NO:411);-   reverse PCR primer 5′-CCACATGTTCCTGCTCTTGTCCTGG-3′ (SEQ ID NO:412).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA43038 sequence which had the    following nucleotide sequence:    hybridization probe-   5′-CGGTAGTGACTGTACTCTAGTCCTGTITACACCCCGTGGTGCCG-3′ (SEQ ID NO:413).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO534 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue (LIB26).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO534 [herein designated as UNQ335(DNA48333-1321)] (SEQ ID NO:409) and the derived protein sequence forPRO534.

The entire nucleotide sequence of UNQ335 (DNA48333-1321) is shown inFIG. 164 (SEQ ID NO:409). Clone UNQ335 (DNA48333-1321) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 87–89 and ending at the stop codon at nucleotidepositions 1167–1169 (FIG. 164). The predicted polypeptide precursor is360 amino acids long (FIG. 165). The full-length PRO534 protein shown inFIG. 165 has an estimated molecular weight of about 39,885 daltons and apI of about 4.79. Clone UNQ335 (DNA48333-1321) has been deposited withATCC on Mar. 26, 1998. It is understood that the deposited clonecontains the actual sequence, and that the sequences provided herein arerepresentative based on current sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO534polypeptide suggests that portions of it possess significant sequenceidentity with the protein disulfide isomerase, thereby indicating thatPRO534 may be a novel disulfide isomerase.

Still analyzing the amino acid sequence of PRO534, the signal peptidesis at about amino acids 1–25 of SEQ ID NO:410. The transmembrane domainis at about amino acids 321–340 of SEQ ID NO:410. The disulfideisomerase corresponding region is at amino acids 212–302 of SEQ IDNO:410. The thioredoxin domain is at amino acids 211–227 of SEQ IDNO:410. N-glycosylation sites are at: 165–168, 181–184, 187–190,194–197, 206–209, 278–281, and 293–296 of SEQ ID NO:410. Thecorresponding nucleotides can routinely be determined from the sequencesprovided herein. PRO534 has a transmembrane domain rather than an ERretention peptide like other protein disulfide isomerases. Additionally,PRO534 may have an intron at the 5 prime end.

Example 66 Isolation of cDNA Clones Encoding Human PRO697

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43052. Based on this consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO697.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CCTGGCTCGCTGCTGCTGCTC-3′ (SEQ ID NO:416);-   reverse PCR primer 5′-CCTCACAGGTGCACTGCAAGCTGTC-3′ (SEQ ID NO:417).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA43052 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-CTCTTCCTCTTTGGCCAGCCCGACTTCTCCTACAAGCGCAGAATTGC-3′ (SEQ ID    NO:418).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO697 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO697 [herein designated as UNQ361(DNA50920-1325)] (SEQ ID NO:414) and the derived protein sequence forPRO697.

The entire nucleotide sequence of UNQ361 (DNA50920-1325) is shown inFIG. 166 (SEQ ID NO:414). Clone UNQ361 (DNA50920-1325) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 4446 and ending at the stop codon at nucleotidepositions 929–931 (FIG. 166). The predicted polypeptide precursor is 295amino acids long (FIG. 167). The full-length PRO697 protein shown inFIG. 167 has an estimated molecular weight of about 33,518 daltons and apI of about 7.74. Clone UNQ361 (DNA50920-1325) was deposited with theATCC on Mar. 26, 1998. It is understood that the deposited clonecontains the actual sequence, and that the sequences provided herein arerepresentative based on current sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO697polypeptide suggests that portions of it possess significant sequenceidentity with sFRPs, thereby indicating that PRO697 may be a novel sFRPfamily member.

Still analyzing the amino acid sequence of PRO697, the signal peptidesis at about amino acids 1–20 of SEQ ID NO:415. The cystein rich domain,having identity with the frizzled N-terminus, is at about amino acids153 of SEQ ID NO:415. The corresponding nucleotides can routinely bedetermined from the sequences provided herein.

Example 67 Isolation of cDNA Clones Encoding Human PRO717

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42829. Based on the DNA42829 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO717.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-AGCTTCTCAGCCCTCCTGGAGCAG-3′ (SEQ ID NO:421);-   reverse PCR primer 5′-CGGGTCAATAAACCTGGACGCTTGG-3′ (SEQ ID NO:422).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA42829 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-TATGTGGACCGGACCAAGCACTTCACTGAGGCCACCAAGATTG-3′ (SEQ ID NO:423).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO717 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO717 [herein designated as UNQ385(DNA50988-1326)] (SEQ ID NO:419) and the derived protein sequence forPRO717.

The entire nucleotide sequence of UNQ385 (DNA50988-1326) is shown inFIG. 168 (SEQ ID NO:419). Clone UNQ385 (DNA50988-1326) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 17–19 and ending at the stop codon at nucleotidepositions 1697–1699 (FIG. 168). The predicted polypeptide precursor is560 amino acids long (FIG. 169). The full-length PRO717 protein shown inFIG. 169 has an estimated molecular weight of about 58,427 daltons and apI of about 6.86. Clone UNQ385 (DNA50988-1326) has been deposited withthe ATCC on Apr. 28, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO717polypeptide suggests that PRO717 may be a novel 12 transmembranereceptor. The reverse complement strand of DNA50988 has a stretch thatmatches identically with human regulatory myosin light strand.

Still analyzing the amino acid sequence of SEQ ID NO:420, transmembranedomains are at about amino acids 30–50, 61–79, 98–112, 126–146, 169–182,201–215, 248–268, 280–300, 318–337, 341–357, 375–387, and 420–441 of SEQID NO:420. N-glycosylation sites are at about amino acids 40–43 and 4346of SEQ ID NO:420. A glycosaminoglycan attachment site is at about aminoacids 468–471 of SEQ ID NO:420. The corresponding nucleotides can beroutinely determined given the sequences provided herein.

Example 68 Isolation of cDNA Clones Encoding Human PRO731

A database was used to search expressed sequence tag (EST) databases.The EST database used herein was the proprietary EST DNA databaseLIFESEQ™, of Incyte Pharmaceuticals, Palo Alto, Calif. Incyte clone2581326 was herein identified and termed DNA42801. Based on the DNA42801sequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO731.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GTAAGCACATGCCTCCAGAGGTGC-3′ (SEQ ID NO:426);-   reverse PCR primer 5′-GTGACGTGGATGCTTGGGATGTTG-3′ (SEQ ID NO:427).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA42801 sequence which had the following    nucleotide sequence:    hybridization probe-   5′-TGGACACCTTCAGTATTGATGCCAAGACAGGCCAGGTCATTCTGCGTCGA-3′ (SEQ ID    NO:428).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO731 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human bone marrow tissue (LIB255). The cDNA librariesused to isolate the cDNA clones were constructed by standard methodsusing commercially available reagents such as those from Invitrogen, SanDiego, Calif. The cDNA was primed with oligo dT containing a NotI site,linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sizedappropriately by gel electrophoresis, and cloned in a definedorientation into a suitable cloning vector (such as pRKB or pRKD; pRK5Bis a precursor of pRK5D that does not contain the Sfil site; see, Holmeset al., Science, 253:1278–1280 (1991)) in the unique XhoI and NotIsites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO731 [herein designated as UNQ395(DNA48331-1329)] (SEQ ID NO:424) and the derived protein sequence forPRO731.

The entire nucleotide sequence of UNQ395 (DNA48331-1329) is shown inFIG. 170 (SEQ ID NO:424). Clone UNQ395 (DNA48331-1329) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 329–331 and ending at the stop codon at nucleotidepositions 3881–3883 (FIG. 170). The predicted polypeptide precursor is1184 amino acids long (FIG. 171). The full-length PRO731 protein shownin FIG. 171 has an estimated molecular weight of about 129,022 daltonsand a pI of about 5.2. Clone UNQ395 (DNA48331-1329) was deposited withthe ATCC on Mar. 31, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO731polypeptide suggests that portions of it possess significant identityand similarity to members of the protocadherin family, therebyindicating that PRO731 may be a novel protocadherin.

Still analyzing the amino acid sequence of SEQ ID NO:425, the putativesignal peptide is at about amino acids 1–13 of SEQ ID NO:425. Thetransmembrane domain is at amino acids 719–739 of SEQ ID NO:425. TheN-glycosylation of SEQ ID NO:425 are as follows: 415–418, 582–586,659–662, 662–665, and 857–860. The cadherin extracellular repeateddomain signatures are at about amino acids (of SEQ ID NO:425): 123–133,232–242, 340–350, 448–458, and 553–563. The corresponding nucleotidescan be routinely determined given the sequences provided herein.

Example 69 Isolation of cDNA Clones Encoding Human PRO218

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA17411. Two proprietary Genentech EST sequenceswere employed in the consensus assembly and are shown in FIGS. 174 and175. Based on the DNA17411 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO218.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-AAGTGGAGCCGGAGCCTTCC-3′ (SEQ ID NO:433);-   reverse PCR primer 5′-TCGTTGTTTATGCAGTAGTCGG-3′ (SEQ ID NO:434).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA17411 sequence which had the    following nucleotide sequence:    hybridization probe-   5′-ATTGTTTAAAGACTATGAGATACGTCAGTATGTTGTACAGG-3′ (SEQ ID NO:435).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO218 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB28).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO218 [herein designated as UNQ192(DNA30867-1335)] (SEQ ID NO:429) and the derived protein sequence forPRO218.

The entire nucleotide sequence of UNQ192 (DNA30867-1335) is shown inFIG. 172 (SEQ ID NO:429). Clone UNQ192 (DNA30867-1335) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 150–152 and ending at the stop codon at nucleotidepositions 1515–1517 (FIG. 172). The predicted polypeptide precursor is455 amino acids long (FIG. 173). The full-length PRO218 protein shown inFIG. 173 has an estimated molecular weight of about 52,917 daltons and apI of about 9.5. Clone UNQ192 (DNA30867-1335) has been deposited withthe ATCC on Apr. 28, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO218polypeptide suggests that PRO218 may be a novel trausmembrane protein.

Still analyzing the amino acid sequence of SEQ ID NO:430, the putativesignal peptide is at about amino acids 1 through 23 of SEQ ID NO:430.Transmembrane domains are potentially at about amino acids 37–55,81–102, 150–168, 288–311, 338–356, 375–398, and 425–444 of SEQ IDNO:430. N-glycosylation sites are at about amino acids 67, 180, and 243of SEQ ID NO:430. Eukaryotic cobalamin-binding protein is at about aminoacids 151–160 of SEQ ID NO:430. The corresponding nucleotides can beroutinely determined given the sequences provided herein.

Example 70 Isolation of cDNA Clones Encoding Human PRO768

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43448. Based on the DNA43448 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO768.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GGCTGACACCGCAGTGCTCTTCAG-3′ (SEQ ID NO:438);-   reverse PCR primer 5′-GCTGCTGGGGACTGCAATGTAGCTG-3′ (SEQ ID NO:439).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA43448 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-CATCCTCCATGTCTCCCATGAGGTCTCTAITGCrCCACGAAGCATC-3′ (SEQ ID    NO:440).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO768 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human bone marrow tissue (LIB255).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO768 [herein designated as UNQ406(DNA55737-1345)] (SEQ ID NO:436) and the derived protein sequence forPRO768.

The entire nucleotide sequence of UNQ406 (DNA55737-1345) is shown inFIG. 176 (SEQ ID NO:436). Clone UNQ406 (DNA55737-1345) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 20–22 and ending at the stop codon at nucleotidepositions 3443–3445 (FIG. 176). The predicted polypeptide precursor is1141 amino acids long (FIG. 177). The full-length PRO768 protein shownin FIG. 177 has an estimated molecular weight of about 124,671 daltonsand a pI of about 5.82. Clone UNQ406 (DNA55737-1345) has been depositedwith the ATCC on Apr. 6, 1998. Regarding the sequence, it is understoodthat the deposited clone contains the correct sequence, and thesequences provided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO768polypeptide suggests that portions of it possess significant sequenceidentity and similarity with integrin 7.

Still analyzing the amino acid sequence of SEQ ID NO:437, the putativesignal peptide is at about amino acids 1–33 of SEQ ID NO:437. Thetransmembrane domain is at amino acids 1039–1064 of SEQ ID NO:437.N-glycosylation sites are at amino acids: 86–89, 746–749, 949–952,985–988 and 1005–1008 of SEQ ID NO:437. Integrin alpha chain proteindomains are identified at about amino acids: 1064–1071, 384–409,1041–1071, 317–346, 443–465, 385–407, 215–224, 634–647, 85–99, 322–346,470–479, 442–466, 379–408 and 1031–1047 of SEQ ID NO:437. Thecorresponding nucleotides can be routinely determined given thesequences provided herein.

Example 71 Isolation of cDNA Clones Encoding Human PRO771

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43330. Based on the DNA43330 sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO771.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CAGCAATATTCAGAAGCGGCAAGGG-3′ (SEQ ID NO:443);-   reverse PCR primer 5′-CATCATGGTCATCACCACCATCATCATC-3′ (SEQ ID    NO:444).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA43330 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-GGTTACTACAAGCCAACACAATGTCATGGCAGTGTTGGACAGTGCTGG-3 ′ (SEQ ID    NO:445).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO771 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB28).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO771 [herein designated as UNQ409(DNA49829-1346)] (SEQ ID NO:441) and the derived protein sequence forPRO771.

The entire nucleotide sequence of UNQ409 (DNA49829-1346) is shown inFIG. 178 (SEQ ID NO:441). Clone UNQ409 (DNA49829-1346) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 134–136 and ending at the stop codon at nucleotidepositions 1442–1444 (FIG. 178). The predicted polypeptide precursor is436 amino acids long (FIG. 179). The full-length PRO771 protein shown inFIG. 179 has an estimated molecular weight of about 49,429 daltons and apI of about 4.8. Clone UNQ409 (DNA49829-1346) has been deposited withthe ATCC on Apr. 7, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO771polypeptide suggests that portions of it possess significant homology tothe testican protein, thereby indicating that PRO771 may be a noveltestican homologue.

Still analyzing the amino acid sequence of SEQ ID NO:442, the putativesignal peptide, leucine zipper pattern, N-myristoylation sites, andthyroglobulin type-1 repeats are also shown in FIG. 179. Thecorresponding nucleotides can be routinely determined given thesequences provided herein.

Example 72 Isolation of cDNA Clones Encoding Human PRO733

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA45600. Based on the DNA45600 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO733.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-CCCAGCAGGGATGGGCGACAAGA-3′ (SEQ ID NO:448);-   reverse PCR primer 5′-GTCTTCCAGTTTCATATCCAATA-3′ (SEQ ID NO:449).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA45600 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-CCAGAAGGAGCACGGGGAAGGGCAGCCAGATCTTGTCGCCCAT-3′ (SEQ ID NO:450).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO733 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human bone marrow tissue (LIB255).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO733 [herein designated as UNQ411(DNA52196-1348)] (SEQ ID NO:446) and the derived protein sequence forPRO733.

The entire nucleotide sequence of UNQ411 (DNA52196-1348) is shown inFIG. 180 (SEQ ID NO:446). Clone UNQ141 (DNA52196-1348) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 106–108 and ending at the stop codon at nucleotidepositions 793–795 (FIG. 180). The predicted polypeptide precursor is 229amino acids long (FIG. 181). The full-length PRO733 protein shown inFIG. 181 has an estimated molecular weight of about 26,017 daltons and apI of about 4.73. Clone UNQ411 (DNA52196-1348) has been deposited withthe ATCC on Apr. 7, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO733polypeptide suggests that portions of it possess significant sequenceidentity and similarity to the T1/ST2 receptor binding protein precursorand therefore may have a similar function in cell signaling. If it is acytokine, it may be useful in the treatment of inflammation and cancer.

Still analyzing the amino acid sequence of SEQ ID NO:447, the putativesignal peptide, transmembrane domain, N-myristoylation site, andtyrosine kinase site are also shown in FIG. 181. The correspondingnucleotides can be routinely determined given the sequences providedherein.

Example 73 Isolation of cDNA Clones Encoding Human PRO162

An expressed sequence tag (EST) DNA database (Merck/WashingtonUniversity) was searched and an EST AA397543 was identified which showedhomology to human pancreatitis-associated protein. The EST AA397543 colewas purchased and its insert obtained and sequenced and the sequenceobtained is shown in FIG. 182 (SEQ ID NO:451).

The entire nucleotide sequence of PRO162 is shown in FIG. 182 (SEQ IDNO:451). DNA sequencing of the clone gave the full-length DNA sequencefor PRO162 [herein designated as UNQ429 (DNA56965-1356)] (SEQ ID NO:451)and the derived protein sequence for PRO162. Clone UNQ429(DNA56965-1356) contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 86–88 and endingat the stop codon at nucleotide positions 611–613 (FIG. 182). Thepredicted polypeptide precursor is 175 amino acids long (FIG. 183). Thefull-length PRO162 protein shown in FIG. 183 has an estimated molecularweight of about 19,330 daltons and a pI of about 7.25. Clone UNQ429(DNA56965-1356) has been deposited with the ATCC. Regarding thesequence, it is understood that the deposited clone contains the correctsequence, and the sequences provided herein are based on knownsequencing techniques.

Analysis of the amino acid sequence of the full-length PRO162polypeptide suggests that portions of it possess significant homology tothe human pancreatitis-associated protein, thereby indicating thatPRO162 may be a novel pancreatitis-associated protein.

Still analyzing the amino acid sequence of SEQ ID NO:452, the putativesignal peptide is at about amino acids 1–26 of SEQ ID NO:452. A C-typelectin domain signature is at about amino acids 146–171 of SEQ IDNO:452. The corresponding nucleotides can be routinely determined giventhe sequences provided herein.

Example 74 Isolation of cDNA Clones Encoding Human PRO788

A consensus DNA sequence (designated herein as DNA49308) was assembledrelative to other EST sequences using phrap as described in Example 1above. Based upon an observed homology between the DNA49308 consensussequence and the Incyte EST clone no. 2777282, the Incyte EST clone no.2777282 was purchased and its insert obtained and sequenced, which gavethe full-length DNA sequence for PRO788 [herein designated as UNQ430(DNA56405-1357)] (SEQ ID NO:453) and the derived protein sequence forPRO788.

Clone UNQ430 (DNA56405-1357) contains a single open reading frame withan apparent translational initiation site at nucleotide positions 84–86and ending at the stop codon at nucleotide positions 459–461 (FIG. 184).The predicted polypeptide precursor is 125 amino acids long (FIG. 185).The full-length PRO788 protein shown in FIG. 185 has an estimatedmolecular weight of about 13,115 daltons and a pI of about 5.90. CloneUNQ430 (DNA56405-1357) has been deposited with the ATCC. Regarding thesequence, it is understood that the deposited clone contains the correctsequence, and the sequences provided herein are based on knownsequencing techniques.

Still analyzing FIG. 185, a signal peptide is shown at about amino acids1–17 of SEQ ID NO:454. An N-glycosylation site is at about amino acids46–49 of SEQ ID NO:454.

Example 75 Isolation of cDNA Clones Encoding Human PRO1008

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated as DNA49804. An EST proprietary to Genentech wasemployed in the consensus assembly and is herein designated as DNA16508(FIG. 188; SEQ ID NO:457). Based upon an observed homology between theDNA49804 sequence and Merck EST clone no. AA143670, the Merck EST cloneno. AA143670 was purchased and its insert obtained and sequenced. Thatsequence is shown herein in FIG. 186 (SEQ ID NO:455).

Sequencing gave the full length sequence for PRO1008 [herein designatedas UNQ492 (DNA57530-1375)] (SEQ ID NO:455) and the derived proteinsequence for PRO1008 were identified.

The entire nucleotide sequence of UNQ492 (DNA57530-1375) is shown inFIG. 186 (SEQ ID NO:455). Clone UNQ492 (DNA57530-1375) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 138–140 and ending at the stop codon at nucleotidepositions 936–938 (FIG. 186). The predicted polypeptide precursor is 266amino acids long (FIG. 187). The full-length PRO1008 protein shown inFIG. 187 has an estimated molecular weight of about 28,672 daltons and apI of about 8.85. Clone UNQ492 (DNA57530-1375) has been deposited withthe ATCC on May 20, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO1008polypeptide suggests that portions of it possess significant sequenceidentity and/or similarity with mdkk-1, thereby indicating that PRO1008may be a novel member of this family and have head inducing activity.

Still analyzing the amino acid sequence of SEQ ID NO:456, the putativesignal peptide is at about amino acids 1–23 of SEQ ID NO:456. TheN-glycosylation site is at about amino acids 256–259 of SEQ ID NO:456,and the fungal zn-(2)-cys(6) binuclear cluster domain is at about aminoacids 110–126 of SEQ ID NO:456. The corresponding nucleotides can of allthe amino acids can be routinely determined given the sequences providedherein.

Example 76 Isolation of cDNA Clones Encoding Human PRO1012

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above, wherein the consensussequence is herein designated DNA49313. Based on the DNA49313 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence forPRO1012.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-ACTCCCCAGGCTGTTCACACTGCC-3′ (SEQ ID NO:460);-   reverse PCR primer 5′-GATCAGCCAGCCAATACCAGCAGC-3′ (SEQ ID NO:461).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA49313 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5′-GTGGTGATGATAGAATGCTTTGCCGAATGAAAGGAGTCAACAGCTATCCC-3′ (SEQ ID    NO:462).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1012 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1012 [herein designated as UNQ495(DNA56439-1376)] (SEQ ID NO:458) and the derived protein sequence forPRO1012.

The entire nucleotide sequence of UNQ495 (DNA56439-1376) is shown inFIG. 189 (SEQ ID NO:458). Clone UNQ495 (DNA56439-1376) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 404–406 and ending at the stop codon at nucleotidepositions 2645–2647 (FIG. 189). The predicted polypeptide precursor is747 amino acids long (FIG. 190). The full-length PRO1012 protein shownin FIG. 190 has an estimated molecular weight of about 86,127 daltonsand a pI of about 7.46. Clone UNQ495 (DNA56439-1376) has been depositedwith ATCC on May 14, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO1012polypeptide suggests that portions of it possess sequence identity withdisulfide isomerase thereby indicating that PRO1012 may be a noveldisulfide isomerase related protein.

Still analyzing the amino acid sequence of SEQ ID NO:459, the cytochromeC family heme-binding site signature is at about amino acids 158–163 ofSEQ ID NO:459. The Nt-DNAJ domain signature is at about amino acids77–96 of SEQ ID NO:459. An N-glycosylation site is at about amino acids484–487 of SEQ ID NO:459. The ER targeting sequence is at about aminoacids 744–747 of SEQ ID NO:459. It is understood that the polypeptideand nucleic acids disclosed can be routinely formed with or without,these portions as desired, in alternative embodiments. For example, itmay be desirable to produce PRO1012 without the ER targeting sequence.The corresponding nucleotides can be routinely determined given thesequences provided herein.

Example 77 Isolation of cDNA Clones Encoding Human PRO1014

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above, wherein the consensussequence obtained is herein designated DNA49811. Based upon an observedhomology between the DNA49811 sequence and Incyte EST clone no. 2612207,Incyte EST clone no. 2612207 was purchased and its insert was obtainedand sequenced, wherein the sequence obtained is shown in FIG. 191 (SEQID NO:463).

DNA sequencing gave the full-length DNA sequence for PRO1014 [hereindesignated as UNQ497 (DNA56409-1377)] (SEQ ID NO:463) and the derivedprotein sequence for PRO1014.

The entire nucleotide sequence of UNQ497 (DNA56409-1377) is shown inFIG. 191 (SEQ ID NO:463). Clone UNQ497 (DNA56409-1377) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 66–68 and ending at the stop codon at nucleotidepositions 966–968 (FIG. 191). The predicted polypeptide precursor is 300amino acids long (FIG. 192). The full-length PRO1014 protein shown inFIG. 192 has an estimated molecular weight of about 33,655 daltons and apI of about 9.31. Clone UNQ497 (DNA56409-1377) has been deposited withthe ATCC on May 20, 1998. Regarding the sequence, it is understood thatthe deposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO1014polypeptide suggests that portions of it possess sequence identity withreductase, thereby indicating that PRO1014 may be a novel member of thereductase family.

Still analyzing the amino acid sequence of SEQ ID NO:464, the putativesignal peptide is at about amino acids 1–19 of SEQ ID NO:464. The cAMPand cGMP dependent protein kinase phosphorylation sites are at aboutamino acids 30–33 and 58–61 of SEQ ID NO:464. Short chain alcoholdehydrogenase family proteins are at about amino acids 165–202, 37–49,112–122 and 210–219 of SEQ ID NO:464. The corresponding nucleotides ofthese domains and any other amino acids provided herein can be routinelydetermined given the sequences provided herein.

Example 78 Isolation of cDNA Clones Encoding Human PRO1017

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above, wherein that consensus DNAsequence is herein designated DNA53235. Based upon an observed homologybetween the DNA53235 consensus sequence and the Merck EST clone no.AA243086, the Merck EST clone no. AA243086 was purchased and its insertobtained and sequenced, wherein the sequence obtained is shown in FIG.193 (SEQ ID NO:465). DNA sequencing gave the full-length DNA sequencefor PRO1017 [herein designated as UNQ500 (DNA56112-1379)] (SEQ IDNO:465) and the derived protein sequence for PRO1017.

The entire nucleotide sequence of UNQ500 (DNA56112-1379) is shown inFIG. 193 (SEQ ID NO:465). Clone UNQ500 (DNA56112-1379) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 128–130 and ending at the stop codon at nucleotidepositions 1370–1372 (FIG. 193). The predicted polypeptide precursor is414 amino acids long (FIG. 194). The full-length PRO1017 protein shownin FIG. 194 has an estimated molecular weight of about 48,414 daltonsand a pI of about 9.54. Clone UNQ500 (DNA56112-1379) has been depositedwith the ATCC. Regarding the sequence, it is understood that thedeposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO1017polypeptide suggests that portions of it possess sequence identity withHNK-1 sulfotransferase, thereby indicating that PRO1017 may be a novelsulfotransferase.

Still analyzing the amino acid sequence of SEQ ID NO:466, the putativesignal peptide is at about amino acids 1–31 of SEQ ID NO:466.N-glycosylation sites are at about amino acids 134–137, 209–212, 280–283and 370–273 of SEQ ID NO:466. The TNFR/NGFR family cystein-rich regionprotein is at about amino acids 329–332 of SEQ ID NO:466. Thecorresponding nucleotides can be routinely determined given thesequences provided herein. The protein can be secreted.

Example 79 Isolation of cDNA Clones Encoding Human PRO474

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above, wherein the consensussequence obtained is herein designated DNA49818. Based upon an observedhomology between the DNA49818 consensus sequence and the Merck EST cloneno. H77889, the Merck EST clone no. H77889 was purchased and its insertobtained and sequenced, wherein the sequence obtained is herein shown inFIG. 195 (SEQ ID NO:467). DNA sequencing gave the full-length DNAsequence for PRO474 [herein designated as UNQ502 (DNA56045-1380)] (SEQID NO:467) and the derived protein sequence for PRO474.

The entire nucleotide sequence of UNQ502 (DNA56045-1380) is shown inFIG. 195 (SEQ ID NO:467). Clone UNQ502 (DNA56045-1380) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 106–108 and ending at the stop codon at nucleotidepositions 916–918 (FIG. 195). The predicted polypeptide precursor is 270amino acids long (FIG. 196). The full-length PRO474 protein shown inFIG. 196 has estimated molecular weight of about 28,317 daltons and a pIof about 6.0. Clone UNQ502 (DNA56045-1380) has been deposited with theATCC. Regarding the sequence, it is understood that the deposited clonecontains the correct sequence, and the sequences provided herein arebased on known sequencing techniques.

Still analyzing the amino acid sequence of SEQ ID NO:468, anN-glycosylation site is at about amino acids 138–141 of SEQ ID NO:468.Short-chain alcohol dehydrogenase family proteins are at about aminoacids 10–22, 81–91, 134–171 and 176–185 of SEQ ID NO:468. Thecorresponding nucleotides can be routinely determined given thesequences provided herein.

Example 80 Isolation of cDNA Clones Encoding Human PRO1031

An initial consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example 1 above, wherein theconsensus sequence obtained is herein designated as DNA47332. Based uponan observed homology between the DNA47332 sequence and the Merck ESTclone no. W74558, Merck EST clone no. W74558 was purchased and itsinsert obtained and sequenced, wherein the sequence obtained is shown inFIG. 197 (SEQ ID NO:469). DNA sequencing gave the full-length DNAsequence for PRO1031 [herein designated as UNQ516 (DNA59294-1381)] (SEQID NO:469) and the derived protein sequence for PRO1031.

The entire nucleotide sequence of UNQ516 (DNA59294-1381) is shown inFIG. 197 (SEQ ID NO:469). Clone UNQ516 DNA59294-1381) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 4244 and ending at the stop codon at nucleotidepositions 582–584 (FIG. 197). The predicted polypeptide precursor is 180amino acids long (FIG. 198). The full-length PRO1031 protein shown inFIG. 198 has an estimated molecular weight of about 20,437 daltons and apI of about 9.58. Clone UNQ516 (DNA59294-1381) has been deposited withthe ATCC. Regarding the sequence, it is understood that the depositedclone contains the correct sequence, and the sequences provided hereinare based on known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO1031polypeptide suggests that it is a novel cytokine.

Still analyzing the amino acid sequence of SEQ ID NO:470, the putativesignal peptide is at about amino acids 1–20 of SEQ ID NO:470. An Nglycosylation site is at about amino acids 75–78 of SEQ ID NO:470. Aregion having sequence identity with IL-17 is at about amino acids96–180. The corresponding nucleotides can be routinely determined giventhe sequences provided herein.

Example 81 Isolation of cDNA Clones Encoding Human PRO938

A consensus DNA sequence was assembled relative other EST sequencesusing phrap as described in Example 1 above, wherein that consensussequence is herein designated DNA49798. Based on the DNA49798 DNAconsensus sequence, oligonucleotides were synthesized: 1) to identify byPCR a cDNA library that contained the sequence of interest, and 2) foruse as probes to isolate a clone of the full-length coding sequence forPRO938.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GTCCAGCCCATGACCGCCTCCAAC-3′ (SEQ ID NO:473)-   reverse PCR primer 5′-CTCTCCTCATCCACACCAGCAGCC-3′ (SEQ ID NO:474)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA49798 sequence which had the followingnucleotide sequence:

hybridization probe

-   5′-GTGGATGCTGAAATTTACGCCCCATGGTGTCCATCCTGCCAGC-3′ (SEQ ID NO:475)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO938 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO938 [herein designated as UNQ475(DNA56433-1406)] (SEQ ID NO:471) and the derived protein sequence forPRO938.

The entire nucleotide sequence of UNQ475 (DNA56433-1406) is shown inFIG. 199 (SEQ ID NO:471). Clone UNQ475 (DNA56433-1406) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 134–136 and ending at the stop codon at nucleotidepositions 1181–1183 (FIG. 199). The predicted polypeptide precursor is349 amino acids long (FIG. 200). The full-length PRO938 protein shown inFIG. 200 has an estimated molecular weight of about 38,952 daltons and apI of about 4.34. Analysis of the full-length PRO938 sequence shown inFIG. 200 (SEQ ID NO:472) evidences the presence of the followingfeatures: a signal peptide from amino 1 to about amino acid 22, atransmembrane domain from about amino acid 191 to about amino acid 211,a potential N-glycosylation site from about amino acid 46 to about aminoacid 49, a region homologous to disulfide isomerase from about aminoacid 56 to about amino acid 72, and a region having sequence identitywith flavodoxin proteins from about amino acid 173 to about amino acid187.

Clone UNQ475 (DNA56433-1406) has been deposited with ATCC on May 15,1998, and is assigned ATCC Accession No. 209857.

Analysis of the amino acid sequence of the full-length PRO938polypeptide suggests that it possesses significant sequence similarityto protein disulfide isomerase, thereby indicating that PRO938 may be anovel protein disulfide isomerase. An analysis of the Dayhoff database(version 35.45 SwissProt 35) evidenced significant homology between thePRO938 amino acid sequence and the following Dayhoff sequences,P_W03626, P_W03627, P_R70491, GARP_PLAFF, XLU85970_(—)1,ACADISPROA_(—)1, IE68_HSVSA, KSU52064_(—)1, U93872_(—)83, P_R97866.

Example 82 Isolation of cDNA Clones Encoding Human PRO1082

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above, wheein the consensussequence is herein designated DNA38097. Based on this consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence forPRO1082.

A set of PCR primers (two forward and one reverse) were synthesized:

-   forward primer 1 5′-GTCCACAGACAGTCATCTCAGGAGCAG-3′ (SEQ ID NO:478);-   forward primer 2 5′-ACAAGTGTCTTCCCAACCTG-3′ (SEQ ID NO:479);-   reverse primer 1 5′-ATCCTCCCAGAGCCATGGTACCTC-3′ (SEQ ID NO:480).    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the DNA38097 consensus sequence which had the    following nucleotide sequence:    hybridization probe-   5 ′-CCAAGGATAGCTGTTGTTTCAGAGAAAGGATCGTGTGCTGCATCTCCTCCT-3′ (SEQ ID    NO:481).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primers identified above. A positive library was then used toisolate clones encoding the PRO1082 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1082 [herein designated as UNQ539(DNA53912-1457)] (SEQ ID NO:476) and the derived protein sequence forPRO1082.

The entire nucleotide sequence of UNQ539 (DNA53912-1457) is shown inFIG. 201 (SEQ ID NO:476). Clone UNQ539 (DNA53912-1457) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 160–162 and ending at the stop codon at nucleotidepositions 763–765 (FIG. 201). The predicted polypeptide precursor is 201amino acids long (FIG. 202). The full-length PRO1082 protein shown inFIG. 202 has an estimated molecular weight of about 22,563 daltons and apI of about 4.87. Clone UNQ539 (DNA53912-1457) has been deposited withthe ATCC. Regarding the sequence, it is understood that the depositedclone contains the correct sequence, and the sequences provided hereinare based on known sequencing techniques.

Still analyzing the amino acid sequence of SEQ ID NO:477, thetransmembrane domain is at about amino acids 45–65 of SEQ ID NO:477. AcAMP- and cGMP-dependent protein kinase phosphorylation site is at aboutamino acids 197–200 of SEQ ID NO:477. N-myristoylation sites are atabout amino acids 3540 and 151–156 of SEQ ID NO:477. The regions whichshare sequence identity with the LDL receptor are at about amino acids34–67 and 70–200 of SEQ ID NO:477. The corresponding nucleotides ofthese amino acid regions and others can be routinely determined giventhe sequences provided herein.

Example 83 Isolation of cDNA Clones Encoding Human PRO1083

A cDNA sequence was identified using the amylase screening techniquedescribed in Example 2 above, wherein that cDNA sequence is designatedherein as DNA24256 (FIG. 205; SEQ ID NO:484). That cDNA sequence wasthen compared and aligned with other known EST sequences as described inExample 1 above to obtain a consensus DNA sequence which is designatedherein as DNA43422. Based on the DNA 43422 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1083.

A pair of PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-GGCATTGGCAGTGCTGGGTG-3′ (SEQ ID NO:485);-   reverse PCR primer 5′-TGGAGGCAGATGCGGCTGGACG-3′ (SEQ ID NO:486).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1083 gene using the reverse PCR primer.RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1083 [herein designated as UNQ540(DNA50921-1458)] (SEQ ID NO:482) and the derived protein sequence forPRO1083.

The entire nucleotide sequence of UNQ540 (DNA50921-1458) is shown inFIG. 203 (SEQ ID NO:482). Clone UNQ540 (DNA50921-1458) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 214–216 and ending at the stop codon at nucleotidepositions 2293–2295 (FIG. 203). The predicted polypeptide precursor is693 amino acids long (FIG. 204). The full-length PRO1083 protein shownin FIG. 204 has an estimated molecular weight of about 77,738 daltonsand a pI of about 8.87. Clone UNQ540 (DNA50921-1458) has been depositedwith the ATCC. Regarding the sequence, it is understood that thedeposited clone contains the correct sequence, and the sequencesprovided herein are based on known sequencing techniques.

Still analyzing the amino acid sequence of SEQ ID NO:483, the putativesignal peptide is at about amino acids 1–25 of SEQ ID NO:483. Thetransmembrane domains are at about amino acids 382–398, 402–420,445–468, 473–491, 519–537, 568–590 and 634–657 of SEQ ID NO:483. Amicrobodies C-terminal targeting signal is at about amino acids 691–693of SEQ ID NO:483. cAMP- and cGMP-dependent protein kinasephosphorylation sites are at about amino acids 198–201 and 370–373 ofSEQ ID NO:483. N-glycosylation sites are at about amino acids 39–42,148–151, 171–174, 234–237, 303–306, 324–227 and 341–344 of SEQ IDNO:483. A G-protein coupled receptor family domain is at about aminoacids 475–504 of SEQ ID NO:483. The corresponding nucleotides can beroutinely determined given the sequences provided herein.

Example 84 Isolation of cDNA Clones Encoding Human PRO200

Probes based on an expressed sequence tag (EST) identified from theIncyte Pharmaceuticals database due to homology with VEGF were used toscreen a cDNA library derived from the human glioma cell line G61. Inparticular, Incyte Clone “INC1302516” was used to generate the followingfour probes:

-   (SEQ ID NO:489) ACTTCTCAGTGTCCATAAGGG;-   (SEQ ID NO:490) GAACTAAAGAGAACCGATACCATTTCTGGCCAGGTTGTC;-   (SEQ ID NO:491) CACCACAGCGTTTAACCAGG; and-   (SEQ ID NO:492) ACAACAGGCACAGTTCCCAC.

Nine positives were identified and characterized. Three clones containedthe fill coding region and were identical in sequence. Partial cloneswere also identified from a fetal lung library and were identical withthe glioma-derived sequence with the exception of one nucleotide changewhich did not alter the encoded amino acid.

Example 85 Expression Constructs for PRO200

For mammalian protein expression, the entire open reading frame (ORF)was cloned into a CMV-based expression vector. An epitope-tag (FLAG,Kodak) and Histidine-tag (His8) were inserted between the ORF and stopcodon. VEGF-E-His8 and VEGF-E-FLAG were transfected into human embryonickidney 293 cells by SuperFect (Qiagen) and pulse-labeled for 3 hourswith [³⁵S]methionine and [³⁵C]cysteine. Both epitope-tagged proteinsco-migrate when 20 microliters of 15-fold concentrated serum-freeconditioned medium were electrophoresed on a polyacrylamide gel (Novex)in sodium dodecyl sulfate sample buffer (SDS-PAGE). The VEGF-E-IgGexpression plasmid was constructed by cloning the ORF in front of thehuman Fc (IgG) sequence.

The VEGF-E-IgG plasmid was co-transfected with Baculogold BaculovirusDNA (Pharmingen) using Lipofectin (GibcoBRL) into 10⁵ Sf9 cells grown inHink's TNM-FH medium (JRH Biosciences) supplemented with 10% fetalbovine serum. Cells were incubated for 5 days at 28° C. The supernatantwas harvested and subsequently used for the first viral amplification byinfecting Sf9 cells at an approximate multiplicity of infection (MOI) of10. Cells were incubated for 3 days, then supernatant harvested, andexpression of the recombinant plasmid determined by binding of 1 ml ofsupernatant to 30 μl of Protein-A Sepharose CL-4B beads (Pharmacia)followed by subsequent SDS-PAGE analysis. The first amplificationsupernatant was used to infect a 500 ml spinner culture of Sf9 cellsgrown in ESF-921 medium (Expression Systems LLC) at an approximate MOIof 0.1. Cells were treated as above, except harvested supernatant wassterile filtered. Specific protein was purified by binding to Protein-ASepharose 4 Fast Flow (Pharmacia) column.

Example 86 Northern Blot Analyses for PRO200

Blots of human poly(A)+ RNA from multiple adult and fetal tissues andtumor cell lines were obtained from Clontech (Palo Alto, Calif.).Hybridization was carried out using ³²P-labeled probes containing theentire coding region and washed in 0.1×SSC, 0.1% SDS at 63° C.

VEGF-E mRNA was detectable in fetal lung, kidney, brain, liver and adultheart, placenta, liver, skeletal muscle, kidney, and pancreas. VEGF-EmRNA was also found in A549 lung adenocarcinoma and HeLa cervicaladenocarcinoma cell lines.

Example 87 In Situ Hybridization of Human Fetal Tissue Sections forPRO200

Formalin-fixed, paraffin-embedded human fetal brain, liver, lower limb,small intestine, thyroid, lymph node, thymus, stomach, trachea, skin,spleen, spinal cord, adrenal, placenta, cord, and adult liver, pancreas,lung, spleen, lymph node, adrenal, heart, aorta, and skin weresectioned, deparaffinized, deproteinated in proteinase K (20 μg/ml) for15 minutes at 37° C., and further processed for in situ hybridization asdescribed by Lu LH and Gillett NA (Cell Vision 1:169–176, 1994). A[α³³-P]UTP-labeled antisense riboprobe was generated from a PCR productof 980 bp (primers GGCGGAATCCAACCTGAGTAG and GCGGCTATCCTCCTGTGCTC, SEQID NOS:493 and494, respectively). The slides were dipped in Kodak NTB2nuclear track emulsion and exposed for 4 weeks.

VEGF-E mRNA expression included localization at the growth plate regionand embracing fetal myocytes.

Example 88 Myocyte Hyertrophy Assay for PRO200

Myocytes from neonatal Harlan Sprague Dawley rat heart ventricle (23days gestation) were plated in duplicate at 75000 cells/ml in a 96-wellplate. Cells were treated for 48h with 2000, 200, 20, or 2 ng/mlVEGF-E-IgG. Myocytes were stained with crystal violet to visualizemorphology and scored on a scale of 3 to 7, 3 being nonstimulated and 7being full-blown hypertrophy.

2000 ng/ml and 200 ng/ml VEGF-E caused hypertrophy, scored as a 5.

Example 89 Cell Proliferation Assay for PRO200

Mouse embryonic fibroblast C3HIOT1/2 cells (ATCC) were grown in 50:50Ham's F-12: low glucose DMEM medium containing 10% fetal calf serum(FCS). Cells were plated in duplicate in a 24-well plate at 1000, 2000,and 4000 cells/well. After 48 hours, cells were switched to mediumcontaining 2% FCS and were incubated for 72 hours with 200, 800, or 2000ng/ml VEGF-E or no growth factor added.

Approximately 1.5 fold greater number of cells were measured in thepresence of 200 ng/ml VEGF-E as in its absence, at all three celldensities.

Example 90 Endothelial Cell Survival Assay for PRO200

Human umbilical vein endothelial cells (HUVEC, Cell Systems) weremaintained in Complete Media (Cell Systems) and plated in triplicate inserum-free medium (Basic Media from Cell Systems containing 0.1% BSA) at20,000 cells/well of a 48-well plate. Cells were incubated for 5 dayswith 200 or 400 ng/ml VEGF-E-IgG, 100 ng/ml VEGF, 20 ng/ml basic FGF, orno addition.

Survival was 2–3 times greater with VEGF-E as compared to lack of growthfactor addition. VEGF and basic FGF were included as positive controls.

Example 91 Isolation of cDNA Clones Encoding Human PRO285

A proprietary expressed sequence tag (EST) DNA database LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST (#2243209)was identified which showed homology to the Drosophila Toll protein.

Based on the EST, a pair of PCR primers (forward and reverse):

-   TAAAGACCCAGCTGTGACCG (SEQ ID NO:499)-   ATCCATGAGCCTGATGGG (SEQ ID NO: 500), and    a probe:-   ATTTATGTCTCGAGGAAAGGGACTGGTTACCAGGGCAGCCAGTTC (SEQ ID NO: 501) were    synthesized.

mRNA for construction of the cDNA libraries was isolated from humanplacenta tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. (Fast Track 2). ThecDNA was primed with oligo dT containing a NotI site, linked with bluntto SalI hemikinased adaptors, cleaved with NotI, sized appropriately bygel electrophoresis, and cloned in a defined orientation into thecloning vector pCR2.1 (Invitrogen, Inc.) using reagents and protocolsfrom Life Technologies, Gaithersburg, Md. (Super Script Plasmid System).The double stranded cDNA was sized to greater than 1000 bp and the cDNAwas cloned into BamHI/NotI cleaved vector. pCR2.1 is a commerciallyavailable plasmid, designed for easy cloning of PCR fragments, thatcarries AmpR and KanR genes for selection, and LacZ gene for blue-whiteselection.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO285 gene using the probe oligonucleotideand one of the PCR primers.

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof DNA40021-1154 (encoding PRO285) is shown in FIG. 208 (SEQ ID NO:495).Clone DNA40021-1154 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 61–63(FIG. 208). The predicted polypeptide precursor is 1049 amino acidslong, including a putative signal peptide at amino acid positions 1–29,a putative transmembrane domain between amino acid positions 837–860,and a leucine zipper pattern at amino acid positions 132–153 and704–725, respectively. It is noted that the indicated boundaries areapproximate, and the actual limits of the indicated regions might differby a few amino acids. Clone DNA40021-1154 has been deposited with ATCC(designation: DNA40021-1154) and is assigned ATCC deposit no. 209389.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence is a human analogue of theDrosophila Toll protein, and is homologous to the following human Tollproteins: Toll (DNAX# HSU88540-1, which is identical with the randomsequenced full-length cDNA #HUMRSC786-1); Tllo2 (DNAX# HSU88878-1);Toll3 (DNAX# HSU88879-1); and Toll4 (DNAX# HSU88880-1).

Example 92 Isolation of cDNA Clones Encodin Human PRO286

A proprietary expressed sequence tag (EST) DNA database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST(#694401) was identified which showed homology to the Drosophila Tollprotein.

Based on the EST, a pair of PCR primers (forward and reverse):

-   GCCGAGACAAAAACGTTCTCC (SEQ ID NO:502)-   CATCCATGTTCTCATCCATTAGCC (SEQ ID NO: 503), and    a probe:-   TCGACAACCTCATGCAGAGCATCAACCAAAGCAAGAAAACAGTATT (SEQ ID NO: 504) were    synthesized.

mRNA for construction of the cDNA libraries was isolated from humanplacenta tissue. This RNA was used to generate an oligo dT primed cDNAlibrary in the vector pRK5D using reagents and protocols from LifeTechnologies, Gaithersburg, Md. (Super Script Plasmid System). pRK5D isa cloning vector that has an sp6 transcription initiation site followedby an SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloningsites. The cDNA was primed with oligo dT containing a NotI site, finkedwith blunt to SalI hemikinased adaptors, cleaved with NotI, sized togreater than 1000 bp appropriately by gel electrophoresis, and cloned ina defined orientation into XhoI/NotI-cleaved pRK5D.

Ea order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO286 gene using the probe oligonucleotideidentified above and one of the PCR primers.

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof DNA42663-1154 (encoding PRO286) shown in FIG. 210 (SEQ ID NO:497).Clone DNA42663-1154 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 57–59(FIG. 211). The predicted polypeptide precursor is 1041 amino acidslong, including a putative signal peptide at amino acid positions 1–26,a potential transmembrane domain at amino acid positions 826–848, andleucine zipper patterns at amino acids 130–151, 206–227, 662–684,669–690 and 693–614, respectively. It is noted that the indicatedboundaries are approximate, and the actual limits of the indicatedregions might differ by a few amino acids. Clone DNA42663-1154 has beendeposited with ATCC (designation: DNA42663-1154) and is assigned ATCCdeposit no. 209386.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence of PRO286, it is a humananalogue of the Drosophila Toll protein, and is homologous to thefollowing human Toll proteins: Toll1 (DNAX# HSU88540-1, which isidentical with the random sequenced full-length cDNA #HUMRSC786-1);Toll2 (DNAX# HSU88878-1); To113 (DNAX# HSU88879-1); and Toll4 (DNAX#HSU88880-1).

Example 93 NF-κB Assay for PRO285 and PRO286

As the Toll proteins signal through the NF-κB pathway, their biologicalactivity can be tested in an NF-κB assay. In this assay Jurkat cells aretransiently transfected using Lipofectamine reagent (Gibco BRL)according to the manufacturer's instructions. 1 μg pB2XLuc plasmid,containing NF-κB driven luciferase gene, is contransfected with 1 μgpSRαN expression vector with or without the insert encoding PRO285 orPRO286. For a positive control, cels are treated with PMA (phorbolmyristyl acetate; 20 ng/ml) and PHA (phytohaemaglutinin, 2 μg/ml) forthree to four hours. Cells are lysed 2 or 3 days later for measurementof luciferase activity reagents from Promega.

Example 94 Isolation of cDNA Clones Encoding Human PRO213-1, PRO1330 andPRO1449

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28735. Based on the DNA28735 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO213-1, PRO1330and/or PRO1449. A pair of PCR primers (forward and reverse) weresynthesized:

-   forward PCR primer 5′-TGGAGCAGCAATATGCCAGCC-3′ (SEQ ID NO:511)-   reverse PCR primer 5′-TTTTCCACTCCTGTCGGGTTGG-3′ (SEQ ID NO:512)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA28735 sequence which had the    following nucleotide sequence:    hybridization probe-   5′-GGTGACACTTGCCAGTCAGATGTGGATGAATGCAGTGCTAGGAGGG-3′ (SEQ ID NO:513)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO213-1, PRO1330 and/or PRO1449 gene usingthe probe oligonucleotide and one of the PCR primers. RNA forconstruction of the cDNA libraries was isolated from human fetal lungtissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence encoding PRO213-1, PRO1330 and/or PRO1449[DNA30943-1-1163-1 (SEQ ID NO:505), DNA64907-1163-1 (SEQ ID NO:507) andDNA64908-1163-1 (SEQ ID NO:509), respectively].

The entire nucleotide sequences corresponding to DNA30943-1-1163-1 (SEQID NO:505), DNA64907-1163-1 (SEQ ID NO:507) and DNA64908-1163-1 (SEQ IDNO:509), respectively. DNA30943-1163, DNA64907-1163-1 andDNA64908-1163-1 contain a single open reading frame with an apparenttranslational initiation site at nucleotide positions 398–401, 488–490and 326–328, respectively, and ending at the stop codon at nucleotidepositions 1221–1223, 1307–1309 and 1145–1147, respectively (FIGS. 212,214 and 216). The predicted polypeptide precursor is 295, 273 and 273amino acids long, respectively (FIGS. 213, 215 and 217).DNA30943-1-1163-1, DNA64907-1163-1 and DNA64908-1163-1 have beendeposited with ATCC and are assigned ATCC deposit no: 209791, 203242 and203243, respectively.

Analysis of the amino acid sequence of the full-length PRO213-1polypeptide suggests that a portion of it possess significant homologyto the human growth arrest-specific gene 6 protein. More specifically,an analysis of the Dayhoff database (version 35.45 SwissProt 35)evidenced significant homology between the PRO213 amino acid sequenceand the following Dayhoff sequences, HSMHC3W5A_(—)6 and B48089.

Additional analysis of the amino acid sequence of the full-lengthPRO1330 and PRO1449 polypeptide indicates significant identity withnotch4. More specifically, an analysis of the Dayhoff database (version35.130 SwissProt 35) evidenced significant identity between PRO1330 andthe following Dayhoff sequences, D86566_(—)1 and NEL_HUMAN.

Example 95 Isolation of cDNA Clones Encoding Human PRO298

A cDNA isolated in the amylase screen described in Example 2 above isherein designated DNA26832 (FIG. 220; SEQ ID NO:516). The sequence ofDNA26832 was then used to search expressed sequence tag (EST) databases.The EST databases included public EST databases (e.g., GenBank) and aproprietary EST database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 (Altshul et al.; Methods in Enzymology 266: 469–480 [1996]).Those comparisons resulting in a BLAST score of 70 (or in some cases 90)or greater that did not encode proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap. A consensus sequence was determined, which was thenextended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using the sources of EST sequencesdiscussed above. The extended assembly sequence was designated DNA35861.Based on the DNA35861 consensus sequence, oligonucleotides weresynthesized: 1) to identity by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence of PRO298. Forward and reverse primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100–1000 bp in length. The probe sequence istypically 40–55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1–1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

PCR primers (forward and reverse) and a hybridization probe weresynthesized:

-   forward PCR primer 1 CAACGTGATTTCAAAGCTGGGCTC (SEQ ID NO:517)-   forward PCR primer 2 GCCTCGTATCAAGAATTTCC (SEQ ID NO:518)-   forward PCR primer 3 AGTGGAAGTCGACCTCCC (SEQ ID NO:519)-   reverse PCR primer 1 CTCACCTGAAATCTCTCATAGCCC (SEQ ID NO:520)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO298 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue (LIB25). The cDNA libraries used to isolated the cDNA cloneswere constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with oligo dT containing a Nod site, linked with blunt to Sailhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the Sfil site; see, Holmes et al., Science,253:1278–1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO298 (herein designated UNQ261(DNA39975-12101) (SEQ ID NO:514), and the derived protein sequence forPRO298 (SEQ ID NO:515).

The entire nucleotide sequence of UNQ261 (DNA39975-1210) is shown inFIG. 218 (SEQ ID NO:514). Clone DNA39975-1210 contains a single openreading frame with an apparent translational initiation site atnucleotide positions 375–377. The predicted polypeptide precursor is 364amino acids long. The protein contains four putative transmembranedomains between amino acid positions 36–55 (type II™), 65–84, 188–208,and 229–245, respectively. A putative N-linked glycosylation site startsat amino acid position 253. In addition, the following features havebeen identified in the protein sequence: cAMP- and cGMP-dependentprotein kinase phosphorylation site, starting at position 8;N-myristoylation sites starting a position 173 and 262, respectively;and a ZP domain between amino acid positions 45–60. Clone DNA39975-1210has been deposited with ATCC (Apr. 21, 1998) and is assigned ATCCdeposit no. 209783.

Example 96 Isolation of cDNA Clones Encoding Human PRO337

A cDNA sequence identified in the amylase screen described in Example 2above is herein designated DNA42301 (FIG. 223, SEQ ID NO:524). TheDNA42301 sequence was then compared to other EST sequences using phrapas described in Example 1 above and a consensus sequence designatedherein as DNA28761 was identified. Based on this consensus sequence,oligonucleotides were synthesized: 1) to identify -by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence. In order to screenseveral libraries for a source of a full-length clone, DNA from thelibraries was screened by PCR amplification with the PCR primer pairidentified above. A positive library was then used to isolate clonesencoding the PRO337 gene using the probe oligonucleotide and one of thePCR primers. RNA for construction of the cDNA libraries was isolatedfrom human fetal brain.

A cDNA clone was sequenced in its entirety. The full length nucleotidesequence of DNA43316-1237 is shown in FIG. 221 (SEQ ID NO:522). CloneDNA43316-1237 contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 134–136 (FIG. 221;SEQ ID NO:522). The predicted polypeptide precursor is 344 amino acidslong. Clone DNA43316-1237 has been deposited with ATCC and is assignedATCC deposit no. 209487.

Based on a BLAST-2 and FastA sequence alignment analysis of thefull-length sequence, PRO337 shows amino acid sequence identity to ratneurotrimin (97%).

Example 97 Isolation of cDNA Clones Encoding Human PRO403

Introduction:

Human thrombopoietin (THPO) is a glycosylated hormone of 352 amino acidsconsisting of two domains. The N-terminal domain, sharing 50% similarityto erythropoietin, is responsible for the biological activity. TheC-terminal region is required for secretion. The gene for thrombopoietin(ThPO) maps to human chromosome 3q27-q8 where the six exons of this genespan 7 kilobase base pairs of genomic DNA (Chang et al., Genomics 26:636–7 (1995); Foster et al., Proc. Natl. Acad. Sci. USA 91: 13023–7(1994); Gurney et al., Blood 85: 981–988 (1995). In order to determinewhether there were any genes encoding THPO homologues located in closeproximity to THPO, genomic DNA fragments from this region wereidentified and sequenced. Three P1 clones and one PAC clones (GenomeSystems Inc., St. Louis, Mo.; cat. Nos. P1-2535 and PAC-6539)encompassing the THPO locus were isolated and a 140 kb region wassequenced using the ordered shotgun strategy (Chenet al., Genomics 17:651–656 (1993)), coupled with a PCR-based gap filling approach. Analysisreveals that the region is gene-rich with four additional genes locatedvery close to THPO: tumor necrosis factor-receptor type 1 associatedprotein 2 (TRAP2) and elongation initiation factor gamma (elF4g,chloride channel 2 (CLCN2) and RNA polymerase II subunit hRPB17. Whileno THPO homolog was found in the region, four novel genes have beenpredicted by computer-assisted gene detection (GRAIL)(Xu et al., Gen.Engin. 16: 241–253 (1994), the presence of CpG islands (Cross, S. andBird, A., Curr. Opin. Genet. & Devel. 5: 109–314 (1995), and homology toknown genes (as detected by WU-BLAST2.0)(Altschul and Gish, MethodsEnzymol. 266: 460–480 (1996).

Procedures:

P1 and PAC Clones:

The initial human P1 clone was isolated from a genomic P1 library(Genome Systems Inc., St. Louis, MO; cat. no.: P1-2535) screened withPCR primers designed from the THPO genomic sequence (A. L. Gurney, etal., Blood 85: 981–88 (1995). PCR primers were designed from the endsequences derived from this P1 clone were then used to screen P1 and PAClibraries (Genome Systems, Cat. Nos.: P1-2535 & PAC-6539) to identifyoverlapping clones (PAC1, p1.t, and P1.u). The 3′-end sequence fromPAC.z was used to define the primers used for the screening of a humanBAC library (Genome Systems Inc., St. Louis, Mo.; Cat. No.: BDTW-4533A).

Ordered Shotgun Strategy:

The Ordered Shotgun Strategy (OSS) (Chen et al., Genomics 17: 651–656(1993)) involves the mapping and sequencing of large genomic DNA cloneswith a hierarchical approach. The P1 or PAC clone was sonicated and thefragments subcloned into lambda vector (λBluestar) (Novagen, Inc.,Madison, Wis.; cat. no. 69242-3). The lambda subclone inserts wereisolated by long-range PCR (Barnes, W. Proc. Natl. Acad. Sci. USA 91:2216–2220 (1994) and the ends sequenced. The lambda-end sequences wereoverlapped to create a partial map of the original clone. Those lambdaclones with overlapping end-sequences were identified, the insetssubcloned into a plasmid vector (pUC18 or pUC19, Hoefer PharmaciaBiotech, Inc., San Francisco, Calif., Cat. Nos. 27-4949-01 and27-4951-01) and the ends of the plasmid subclones were sequenced andassembled to generate a contiguous sequence. This directed sequencingstrategy minimizes the redundancy required while allowing one to scanfor and concentrate on interesting regions.

In order to define better the THPO locus and to search for other genesrelated to the hematopoietin family, five genomic clones were isolatedfrom this region by PCR screening of human P1 and PAC libraries (GenomeSystem, Inc., Cat. Nos.: P1-2535 and PAC-6539). The sizes of the genomicfragments are as follows: P1.t is 40 kb; P1.g is 70 kb; P1.u is 70 kb;PAC.z is 200 kb; and BAC.1 is 80 kb. Approximately 75% (140 kb) of the190 kb genomic DNA region was sequenced by the Ordered Shotgun Strategy(OSS) (Chen et al., Genomics 17: 651–56 (1993), and assembled intocontigs using AutoAssembler™ (Applied Biosystems, Perkin Elmer, FosterCity, Calif., cat. no. 903227). The preliminary order of these contigswas determined by manual analysis. There were 47 contigs the 140 kbregion. A PCR-based approach to ordering the contigs and filling in thegaps was employed. The following summarizes the number and sizes of thegaps. The 50 kb of sequence unique to BAC.1 was sequenced by a totalshotgun approach with a ten-fold redundancy.

Size of gap number <50 bp 13 50–150 bp  7 150–300 bp  7 300–1000 bp 101000–5000 bp  7 >5000 bp  2 ((15,000 bp)DNA Sequencing:

ABI DYE-primer™ chemistry (PE Applied Biosystems, Foster City, Calif.;Cat. No.: 402112) was used to end-sequence the lambda and plasmidsubclones. ABI DYE-terminater™ chemistry (PE Applied Biosystems, FosterCity, Calif., Cat. No: 403044) was used to sequence the PCR productswith their respective PCR primers. The sequences were collected with anABI377 instrument. For PCR products larger than 1 kb, walking primerswere used. The sequences of contigs generated by the OSS strategy inAutoAssembler™ (PE Applied Biosystems, Foster City, Calif.; Cat. No:903227) and the gap-filling sequencing trace files were imported intoSequencher™ (Gene Codes Corp., Ann Arbor, Mich.) for overlapping andediting. The sequences generated by the total shotgun strategy wereassembled using Phred and Phrap and edited using Consed and GFP (GenomeReconstruction Manager for Phrap), version 1.2.

PCR-Based Gap Filling Strategy:

Primers were designed based on the 5′- and 3′-end sequenced of eachcontig, avoiding repetitive and low quality sequence regions. Allprimers were designed to be 19–24-mers with 50–70% G/C content. Oligoswere synthesized and gel-purified by standard methods.

Since the orientation and order of the contigs were unknown,permutations of the primers were used in the amplification reactions.Two PCR kits were used: first, XL PCR kit (Perkin Elmer, Norwalk, Conn.;Cat. No.: N8080205), with extension times of approximately 10 minutes;and second, the Taq polymerase PCR kit (Qiagen Inc., Valencia, Calif.;Cat. No.: 201223) was used under high stringency conditions if smearedor multiple products were observed with the XL PCR kit. The main PCRproduct from each successful reaction was extracted from a 0.9% lowmelting agarose gel and purified with the Geneclean DNA Purification kitprior to sequencing.

Analysis:

The identification and characterization of coding regions was carriedout as follows: First, repetitive sequences were masked usingRepeatMasker (A. F. A. Smit & P. Green which screens DNA sequences inFastA format against a library of repetitive elements and returns amasked query sequence. Repeats not masked were identified by comparingthe sequence to the GenBank database using WUBLAST2.0 [Altschul, S &Gish, W., Methods Enzymol. 266: 460–480 (1996)] and were maskedmanually.

Next, known genes were revealed by comparing the genomic regions againstGenentech's protein database using the WUBLAST2.0 algorithm and thenannotated by aligning the genomic and cDNA sequences for each gene,respectively, using a Needleman-Wunch (Needleman and Wunsch, J. Mol.Biol. 48: 443–453 (1970) algorithm to find regions of local identitybetween sequences. The strategy results in detection of all exons of thefive known genes in the region, THPO, TRAP2, eIF4g, CLCN2 and hRPB17(see below).

Known genes Map position eukaryotic translation initiation factor 4gamma 3q27-qter thrombopoietin 3q26-q27 chloride channel 2 3q26-qter TNFreceptor associated protein 2 not previously mapped RNA polymerase IIsubunit hRPB17 not previously mapped

Finally, novel transcription units were predicted using a number ofapproaches. CpG islands (S. Cross & Bird, A., Curr. Opin. Genet. Dev. 5:109–314 (1995) islands were used to define promoter regions and wereidentified as clusters of sites cleaved by enzymes recognizing GC-rich,6 or 8-mer palindromic sequences (NotI, NarI, BssHII, XhoI. CpG islandsare usually associated with promoter regions of genes. WUBLAST2.0analysis of short genomic regions (10–20 kb) versus GenBank revealedmatches to ESTs. The individual EST sequences (or where possible, theirsequence chromatogram files) were retrieved and assembled with Sequencerto provide a theoretical cDNA sequence (DNA36443). GRAIL2 (ApoCom Inc.,Knoxville, Tenn., command line version for the DEC alpha) was used topredict a novel exon. The five known genes in the region served asinternal controls for the success of the GRAIL algorithm.

Isolation:

A partial endothelin converting enzyme-2 (ECE-2) cDNA clone was isolatedby first splicing in silico the ECE-2 exons predicted in the genomicsequence to generate a putative sequence (DNA36443). An oligonucleotideprobe: GAAGCAGTGCAGCCAGCAGTAGAGAGGCACCTGCTAAGA) (SEQ ID NO:530) wasdesigned and used to screen a human fetal small intestine library(LIB110) and internal PCR primers (36443f1)(ECE2.f:ACGCAGCTGGAGCTGGTCTTAGCA) (SEQ ID NO:531) and (36443r1) (ECE2.r)(GGTACTGGACCCCTAGGGCCACAA) (SEQ ID NO:532) were used to confirm cloneshybridizing to the probe prior to sequencing. One positive clone wasobtained, however this cDNA (DNA49830) represented a partially splicedtranscript containing appropriately spliced exons 1 through 6, followedby intron 6 sequence. The oligo dT primer annealed to a polyA-stretchwithin an Alu element present in intron 6. An additional ECE-2 cDNAfragment (DNA49831) was obtained by PCR from a human fetal kidneylibrary (LIB227) with primers designed from the presumed cDNA sequence[36443f3: CCTCCCAGCCGAGACCAGTGG (SEQ ID NO:533) and 36443r2:GGTCCTATAAGGGCCAAGACC (SEQ ID NO:534)]. This PCR product extended fromexon 13 into the 3′ untranslated region in exon 18.

A full length endothelin converting enzyme 2 (ECE-2) cDNA clone(DNA55800-1263) was isolated from an oligo-dT-primed human fetal brainlibrary. RNA from human fetal brain tissue (20 weeks gestation,#283005)(SRC175) was isolated by guanidine thiocyanate and 5 μg used togenerate double stranded cDNA which was cloned into the vector pRK5E.The 3′-primer (pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTT) (SEQ IDNO:535) and the 5-linker (pCGGACGCGTGGGTCGA) (SEQ ID NO:536) weredesigned to introduce XhoI and NotI restriction sites. The library wasscreened with PCR primers [36443pcrf1: CGGCCGTGATGGCTGGTGACG (SEQ IDNO:537) and 36443r3: GGCAGACTCCTTCCTATGGG (SEQ ID NO:538)] designed fromthe partial human ECE-2 cDNA sequences (DNA49830 and DNA49831). PCRproducts were cloned into the vector pCR2.1-TOPO (Invitrogen Corp.,Carlsbad, Calif., Cat. No. K4500-01) and sequenced with DYE-terminatorchemistry as described above.

Example 98 Northern Blot and In Situ RNA Hybridization Analysis forPRO403

Expression of PRO403 mRNA in human tissues was examined by Northern blotanalysis. Human polyA+ RNA blots derived from human fetal and adulttissues (Clontech, Palo Alto, Calif.; Cat. Nos. 7760-1, 7756-1 and7755-1) were hybridized to a [32P-α]dATP-labelled cDNA fragments fromprobe based on the full length PRO403 cDNA. Blots were incubated withthe probes in hybridization buffer (5×SSPE; 2× Denhardt's solution; 100mg/mL denatured sheared salmon sperm DNA; 50% formamide; 2% SDS) for 18hours at 42° C., washed to high stringency (0.1×SSC, 0.1% SDS, 50° C.)and autoradiographed. The blots were developed after overnight exposureby phosphorimager analysis (Fuji).

PRO403 mRNA transcripts were detected. Analysis of the expressionpattern showed the strongest signal of the expected 3.3 kb transcript inadult brain (highest in the cerebellum, putamen, medulla, and temporallobe, and lower in the cerebral cortex, occipital lobe and frontallobe), spinal cord, lung and pancreas and higher levels of a 4.5 kbtranscript in fetal brain and kidney.

Example 99 Use of PRO Polypeptide-Encoding Nucleic Acid as HybridizationProbes

The following method describes use of a nucleotide sequence encoding aPRO polypeptide as a hybridization probe.

DNA comprising the coding sequence of of a PRO polypeptide of interestas disclosed herein may be employed as a probe or used as a basis fromwhich to prepare probes to screen for homologous DNAs (such as thoseencoding naturally-occurring variants of the PRO polypeptide) in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO polypeptide-encoding nucleic acid-derived probe tothe filters is performed in a solution of 50% formamide, 5×SSC, 0.1%SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours.Washing of the filters is performed in an aqueous solution of 0.1×SSCand 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO polypeptide can then be identified usingstandard techniques known in the art.

Example 100 Expression of PRO Polypeptides in E. coli

This example illustrates preparation of an unglycosylated form of adesired PRO polypeptide by recombinant expression in E. coli.

The DNA sequence encoding the desired PRO polypeptide is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the specific PRO polypeptidecoding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO polypeptide can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO181, PRO195, PRO200, PRO237, PRO273, PRO540, PRO322, PRO1017, PRO938,PRO162, PRO114, PRO827 and PRO1008 were expressed in E. coli in apoly-His tagged form, using the following procedure. The DNA encodingthe PRO polypeptide was initially amplified using selected PCR primers.The primers contained restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector, and otheruseful sequences providing for efficient and reliable translationinitiation, rapid purification on a metal chelation column, andproteolytic removal with enterokinase. The PCR-amplified, poly-Histagged sequences were then ligated into an expression vector, which wasused to transform an E. coli host based on strain 52 (W3110 fuhA (tonA)lon galE rpoHts(htpRts) clpP(lacIq). Transformants were first grown inLB containing 50 mg/ml carbenicillin at 30° C. with shaking until anO.D.600 of 3–5 was reached. Cultures were then diluted 50–100 fold intoCRAP media (prepared by mixing 3.57 g (NH₄)₂SO₄, 0.71 g sodiumcitrate.2H₂O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffieldhycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v)glucose and 7 mM MgSO₄) and grown for approximately 20–30 hours at 30°C. with shaking. Samples were removed to verify expression by SDS-PAGEanalysis, and the bulk culture is centrifuged to pellet the cells. Cellpellets were frozen until purification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6–10 g pellets) wasresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionwas stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution wascentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant was diluted with 3–5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. Depending the clarified extract was loaded onto a Sml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelatecolumn buffer. The column was washed with additional buffer containing50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein waseluted with buffer containing 250 mM imidazole. Fractions containing thedesired protein were pooled and stored at 4° C. Protein concentrationwas estimated by its absorbance at 280 nm using the calculatedextinction coefficient based on its amino acid sequence.

The proteins were refolded by diluting sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes were chosen so that the final protein concentration was between50 to 100 micrograms/ml. The refolding solution was stirred gently at 4°C. for 12–36 hours. The refolding reaction was quenched by the additionof TFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution was filtered through a0.22 micron filter and acetonitrile was added to 2–10% finalconcentration. The refolded protein was chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance were analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein were pooled.Generally, the properly refolded species of most proteins are eluted atthe lowest concentrations of acetonitrile since those species are themost compact with their hydrophobic interiors shielded from interactionwith the reversed phase resin. Aggregated species are usually eluted athigher acetonitrile concentrations. In addition to resolving misfoldedforms of proteins from the desired form, the reversed phase step alsoremoves endotoxin from the samples.

Fractions containing the desired folded PRO proteins were pooled and theacetonitrile removed using a gentle stream of nitrogen directed at thesolution. Proteins were formulated into 20 mM Hepes, pH 6.8 with 0.14 Msodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Many of the PRO polypeptides described herein were successfullyexpressed as described above.

Example 101 Expression of PRO Polypeptides in Mammalian Cells

This example illustrates preparation of a glycosylated form of a desiredPRO polypeptide by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO polypeptide-encoding DNAis ligated into pRK5 with selected restriction enzymes to allowinsertion of the PRO polypeptide DNA using ligation methods such asdescribed in Sambrook et al., supra. The resulting vector is calledpRK5-PRO polypeptide.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PROpolypeptide DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, PRO polypeptide may be introduced into 293cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 μg pRK5-PROpolypeptide DNA is added. The cells are first concentrated from thespinner flask by centrifugation and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet for four hours. The cellsare treated with 20% glycerol for 90 seconds, washed with tissue culturemedium, and re-introduced into the spinner flask containing tissueculture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin.After about four days, the conditioned media is centrifuged and filteredto remove cells and debris. The sample containing expressed PROpolypeptide can then be concentrated and purified by any selectedmethod, such as dialysis and/or column chromatography.

In another embodiment, PRO polypeptides can be expressed in CHO cells.The pRK5-PRO polypeptide can be transfected into CHO cells using knownreagents such as CaPO₄ or DEAE-dextran. As described above, the cellcultures can be incubated, and the medium replaced with culture medium(alone) or medium containing a radiolabel such as ³⁵S-methionine. Afterdetermining the presence of PRO polypeptide, the culture medium may bereplaced with serum free medium. Preferably, the cultures are incubatedfor about 6 days, and then the conditioned medium is harvested. Themedium containing the expressed PRO polypeptide can then be concentratedand purified by any selected method.

Epitope-tagged PRO polypeptide may also be expressed in host CHO cells.The PRO polypeptide may be subcloned out of the pRK5 vector. Thesubclone insert can undergo PCR to fuse in frame with a selected epitopetag such as a poly-his tag into a Baculovirus expression vector. Thepoly-his tagged PRO polypeptide insert can then be subcloned into a SV40driven vector containing a selection marker such as DHFR for selectionof stable clones. Finally, the CHO cells can be transfected (asdescribed above) with the SV40 driven vector. Labeling may be performed,as described above, to verify expression. The culture medium containingthe expressed poly-His tagged PRO polypeptide can then be concentratedand purified by any selected method, such as by Ni²⁺-chelate affinitychromatography.

Stable expression in CHO cells was performed using the followingprocedure. The proteins were expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins were fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs were subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24: 9 (1774–1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA were introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Eugene®(Boehringer Mannheim). The cells were grown and described in Lucas etal., supra. Approximately 3×10⁻⁷ cells are frozen in an ampule forfurther growth and production as described below.

The ampules containing the plasmid DNA were thawed by placement intowater bath and mixed by vortexing. The contents were pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant was aspirated and the cells wereresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells were then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1–2days, the cells were transferred into a 250 mL spinner fled with 150 mLselective growth medium and incubated at 37° C. After another 2–3 days,a 250 mL, 500 mL and 2000 mL spinners were seeded with 3×10⁵ cells/mL.The cell media was exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 was actually used. 3 L production spinner is seededat 1.2×10⁶ cells/mL. On day 0, the cell number pH were determined. Onday 1, the spinner was sampled and sparging with filtered air wascommenced. On day 2, the spinner was sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion). Throughout the production, pH was adjusted as necessary tokeep at around 7.2. After 10 days, or until viability dropped below 70%,the cell culture was harvested by centrifugtion and filtering through a0.22 μm filter. The filtrate was either stored at 4° C. or immediatelyloaded onto columns for purification.

For the poly-His tagged constructs, the proteins were purified using aNi-NTA column (Qiagen). Before purification, imidazole was added to theconditioned media to a concentration of 5 mM. The conditioned media waspumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4–5ml/min. at 4° C. After loading, the column was washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein wassubsequently desalted into a storage buffer containing 10 mM Hepes, 0.14M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of were purified from theconditioned media as follows. The conditioned medium was pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity was assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Many of the PRO polypeptides described herein were successfullyexpressed as described above.

Example 102 Expression of PRO Polypeptides in Yeast

The following method describes recombinant expression of a desired PROpolypeptide in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO polypeptides from the ADH2/GAPDHpromoter. DNA encoding a desired PRO polypeptide, a selected signalpeptide and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression of thePRO polypeptide. For secretion, DNA encoding the PRO polypeptide can becloned into the selected plasmid, together with DNA encoding theADH2/GAPDH promoter, the yeast alpha-factor secretory signal/leadersequence, and linker sequences (if needed) for expression of the PROpolypeptide.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO polypeptide can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing the PRO polypeptide may further be purified usingselected column chromatography resins.

Many of the PRO polypeptides described herein were successfullyexpressed as described above.

Example 103 Expression of PRO Polypeptides in Baculovirus-InfectedInsect Cells

The following method describes recombinant expression of PROpolypeptides in Baculovirus-infected insect cells.

The desired PRO polypeptide is fused upstream of an epitope tagcontained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO polypeptide or the desired portion of the PRO polypeptide (such asthe sequence encoding the extracellular domain of a transmembraneprotein) is amplified by PCR with primers complementary to the 5′ and 3′regions. The 5′ primer may incorporate flanking (selected) restrictionenzyme sites. The product is then digested with those selectedrestriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4–5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO polypeptide can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175–179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A280baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO polypeptide are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PROpolypeptide can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

PRO195, PRO526, PRO540, PRO846, PRO362, PRO363, PRO700, PRO707, PRO322,PRO719, PRO1083, PRO868, PRO866, PRO768, PRO788, PRO938, PRO827 andPRO1031 were successfully expressed in baculovirus infected Sf9 insectcells. While the expression was actually performed in a 0.5–2 L scale,it can be readily scaled up for larger (e.g. 8 L) preparations. Theproteins were expressed as an IgG construct (immunoadhesin), in whichthe protein extracellular region was fused to an IgG1 constant regionsequence containing the hinge, CH2 and CH3 domains and/or in poly-Histagged forms.

For expression in baculovirus infected Sf9 cells, following PCRamplification, the respective coding sequences were subcloned into abaculovirus expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.cfor poly-His tagged proteins), and the vector and Baculogold®baculovirus DNA (Pharmingen) were co-transfected into 105 Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711), using Lipofectin (Gibco BRL).pb.PH.IgG and pb.PH.His are modifications of the commercially availablebaculovirus expression vector pVL1393 (Pharmingen), with modifiedpolylinker regions to include the His or Fc tag sequences. The cellswere grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone).Cells were incubated for 5 days at 28° C. The supernatant was harvestedand subsequently used for the first viral amplification by infecting Sf9cells in Hink's TNM-FH medium supplemented with 10% FBS at anapproximate multiplicity of infection (MOI) of 10. Cells were incubatedfor 3 days at 28° C. The supernatant was harvested and the expression ofthe constructs in the baculovirus expression vector was determined bybatch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads (QIAGEN)for histidine tagged proteins or Protein-A Sepharose CL-4B beads(Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysiscomparing to a known concentration of protein standard by Coomassie bluestaining.

The first viral amplification supernatant was used to infect a spinnerculture (500 ml) of Sf9 cells grown in ESF-921 medium (ExpressionSystems LLC) at an approximate MOI of 0.1. Cells were incubated for 3days at 28° C. The supernatant was harvested and filtered. Batch bindingand SDS-PAGE analysis was repeated, as necessary, until expression ofthe spinner culture was confirmed.

The conditioned medium from the transfected cells (0.5 to 3 L) washarvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct were purified using a Ni-NTA column (Qiagen). Beforepurification, imidazole was added to the conditioned media to aconcentration of 5 mM. The conditioned media were pumped onto a 6 mlNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4–5 ml/min. at 4° C. Afterloading, the column was washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein was subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of proteins were purified fromthe conditioned media as follows. The conditioned media were pumped ontoa 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 mL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of the proteins was verified by SDS polyacrylamide gel (PEG)electrophoresis and N-terminal amino acid sequencing by Edmandegradation.

PRO181, PRO195, PRO200, PRO320, PRO237, PRO273, PRO285, PRO337, PRO526,PRO540, PRO846, PRO362, PRO363, PRO617, PRO322, PRO1083, PRO868, 768,PRO792, PRO788, PRO162, PRO1114, PRO827, PRO1075 and PRO1031 weresuccessfully expressed in baculovirus infected Hi5 insect cells. Whilethe expression was actually performed in a 0.5–2 L scale, it can bereadily scaled up for larger (e.g. 8 L) preparations.

For expression in baculovirus-infected Hi5 insect cells, the PROpolypeptide-encoding DNA may be amplified with suitable systems, such asPfu (Stratagene), or fused upstream (5′-of) of an epitope tag containedwith a baculovirus expression vector. Such epitope tags include poly-histags and immunoglobulin tags (like Fe regions of IgG). A variety ofplasmids may be employed, including plasmids derived from commerciallyavailable plasmids such as pVL1393 (Novagen). Briefly, the PROpolypeptide or the desired portion of the PRO polypeptide (such as thesequence encoding the extracellular domain of a transmembrane protein)is amplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector. For example,derivatives of pVL1393 can include the Fc region of human IgG(pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3′-of) theNAME sequence. Preferably, the vector construct is sequenced forconfirmation.

Hi5 cells are grown to a confluency of 50% under the conditions of, 27°C., no CO2, NO pen/strep. For each 150 mm plate, 30 ug of pIE basedvector containing PRO polypeptide is mixed with 1 ml Ex-Cell medium(Media: Ex-Cell 401+1/100 L-Glu JRH Biosciences #14401-78P (note: thismedia is light sensitive)), and in a separate tube, 100 ul of Cellfectin(CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) is mixed with 1 mlof Ex-Cell medium. The two solutions are combined and allowed toincubate at room temperature for 15 minutes. 8 ml of Ex-Cell media isadded to the 2 ml of DNA/CellFECTIN mix and this is layered on Hi5 cellsthat have been washed once with Ex-Cell media. The plate is thenincubated in darkness for 1 hour at room temperature. The DNA/CellFECTINmix is then aspirated, and the cells are washed once with Ex-Cell toremove excess CellFECTIN. 30 ml of fresh Ex-Cell media is added and thecells are incubated for 3 days at 28° C. The supernatant is harvestedand the expression of the PRO polypeptide in the baculovirus expressionvector can be determined by batch binding of 1 ml of supernatent to 25mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-ASepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed bySDS-PAGE analysis comparing to a known concentration of protein standardby Coomassie blue staining.

The conditioned media from the transfected cells (0.5 to 3 L) isharvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteincomprising the PRO polypeptide is purified using a Ni-NTA column(Qiagen). Before purification, imidazole is added to the conditionedmedia to a concentration of 5 mM. The conditioned media is pumped onto a6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffercontaining 0.3 M NaCl and 5 mM imidazole at a flow rate of 4–5 ml/min.at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydeslated into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc containing) constructs of proteins are purified fromthe conditioned media as follows. The conditioned media is pumped onto a5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 mL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of PRO polypeptide can be assessed by SDS polyacrylamidegels and by N-terminal amino acid sequencing by Edman degradation andother analytical procedures as desired or necessary.

Many of the PRO polypeptides described herein were successfullyexpressed as described above.

Example 104 Preparation of Antibodies that Bind to PRO Polypeptides

This example illustrates preparation of monoclonal antibodies which canspecifically bind to a PRO polypeptide.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO polypeptide, fusion proteins containingthe PRO polypeptide, and cells expressing recombinant PRO polypeptide onthe cell surface. Selection of the immunogen can be made by the skilledartisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO polypeptide immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneaUy in an amount from 1–100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mon.) and injected into the animal's hind foot pads.The immunized mice are then boosted 10 to 12 days later with additionalimmunogen emulsified in the selected adjuvant. Thereafter, for severalweeks, the mice may also be boosted with additional immunizationinjections. Serum samples may be periodically obtained from the mice byretro-orbital bleeding for testing in ELISA assays to detect anti-PROpolypeptide antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO polypeptide. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstthe PRO polypeptide. Determination of “positive” hybridoma cellssecreting the desired monoclonal antibodies against the PRO polypeptideis within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PROpolypeptide monoclonal antibodies. Alternatively, the hybridoma cellscan be grown in tissue culture flasks or roller bottles. Purification ofthe monoclonal antibodies produced in the ascites can be accomplishedusing ammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 105 Chimeric PRO Polypeptides

PRO polypeptides may be expressed as chimeric proteins with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS™ extension/affinity purification system (Immunex Corp.,Seattle Wash.). The inclusion of a cleavable linker sequence such asFactor XA or enterokinase (Invitrogen, San Diego Calif.) between thepurification domain and the PRO polypeptide sequence may be useful tofacilitate expression of DNA encoding the PRO polypeptide.

Example 106 Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2–3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 107 Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cels, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (I) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 108 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (cf., Hodgson, Bio/Technology, 9: 19–21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796–7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem. 113:742–746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the ant-ids would beexpected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Example 109 Ability of PRO Polypeptides to Inhibit Vascular EndothelialGrowth Factor (VEGF) Stimulated Proliferation of Endothelial Cell Growth(Assay 9)

The ability of various PRO polypeptides to inhibit VEGF stimulatedproliferation of endothelial cells was tested. Polypeptides testingpositive in this assay are useful for inhibiting endothelial cell growthin mammals where such an effect would be beneficial, e.g., forinhibiting tumor growth.

Specifically, bovine adrenal cortical capillary endothelial cells (ACE)(from primary culture, maximum of 12–14 passages) were plated in 96-wellplates at 500 cells/well per 100 microliter. Assay media included lowglucose DMEM, 10% calf serum, 2 mM glutamine, and 1×penicillin/streptomycin/fungizone. Control wells included the following:(1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/mlFGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGFplus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus 5 ng/mlLIF. The test samples, poly-his tagged PRO polypeptides (in 100microliter volumes), were then added to the wells (at dilutions of 1%,0.1% and 0.01%, respectively). The cell cultures were incubated for 6–7days at 37° C./5% CO₂. After the incubation, the media in the wells wasaspirated, and the cells were washed 1× with PBS. An acid phosphatasereaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1%Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to eachwell. After a 2 hour incubation at 37° C., the reaction was stopped byaddition of 10 microliters 1N NaOH. Optical density (OD) was measured ona microplate reader at 405 nm.

The activity of PRO polypeptides was calculated as the percentinhibition of VEGF (3 ng/ml) stimulated proliferation (as determined bymeasuring acid phosphatase activity at OD 405 nm) relative to the cellswithout stimulation. TGF-beta was employed as an activity reference at 1nglml, since TGF-beta blocks 70–90% of VEGF-stimulated ACE cellproliferation. The results are indicative of the utility of the PROpolypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of VEGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation and thendividing that percentage into the percent inhibition obtained by TGF-βat 1 ng/ml which is known to block 70–90% of VEGF stimulated cellproliferation. The results are considered positive if the PROpolypeptide exhibits 30% or greater inhibition of VEGF stimulation ofendothelial cell growth (relative inhibition 30% or greater).

The following polypeptides tested positive in this assay: PRO200, PRO322and PRO320.

Example 110 Retinal Neuron Survival (Assay 52)

This example demonstrates that certain PRO polypeptides have efficacy inenhancing the survival of retinal neuron cells and, therefore, areuseful for the therapeutic treatment of retinal disorders or injuriesincluding, for example, treating sight loss in mammals due to retinitispigmentosum, AMD, etc.

Sprague Dawley rat pups at postnatal day 7 (mixed population: glia andretinal neuronal types) are killed by decapitation following CO₂anesthesia and the eyes are removed under sterile conditions. The neuralretina is dissected away from the pigment epithelium and other oculartissue and then dissociated into a single cell suspension using 0.25%trypsin in Ca²⁺, Mg²⁺-free PBS. The retinas are incubated at 37° C. for7–10 minutes after which the trypsin is inactivated by adding 1 mlsoybean trypsin inhibitor. The cells are plated at 100,000 cells perwell in 96 well plates in DMEM/F12 supplemented with N2 and with orwithout the specific test PRO polypeptide. Cells for all experiments aregrown at 37° C. in a water saturated atmosphere of 5% CO₂. After 2–3days in culture, cells are stained with calcein AM then fixed using 4%paraformaldehyde and stained with DAPI for determination of total cellcount. The total cells (fluorescent) are quantified at 20× objectivemagnification using CCD camera and NIH image software for MacIntosh.Fields in the well are chosen at random.

The effect of various concentration of PRO polypeptides are reportedherein where percent survival is calculated by dividing the total numberof calcein AM positive cells at 2–3 days in culture by the total numberof DAPI-labeled cells at 2–3 days in culture. Anything above 30%survival is considered positive.

The following PRO polypeptides tested positive in this assay usingpolypeptide concentrations within the range of 0.01% to 1.0% in theassay: PRO200, PRO322, PRO540, PRO846 and PRO617.

Example 111 Rod Photoreceptor Survival (Assay 56)

This assay shows that certain polypeptides of the invention act toenhance the survival/proliferation of rod photoreceptor cells and,therefore, are useful for the therapeutic treatment of retinal disordersor injuries including, for example, treating sight loss in mammals dueto retinitis pigmentosum, AMD, etc. Sprague Dawley rat pups at 7 daypostnatal (mixed population: glia and retinal neuronal cell types) arekilled by decapitation following CO₂ anesthesis and the eyes are removedunder sterile conditions. The neural retina is dissected away form thepigment epithelium and other ocular tissue and then dissociated into asingle cell suspension using 0.25% trypsin in Ca²⁺, Mg²⁺-free PBS. Theretinas are incubated at 37° C. for 7–10 minutes after which the trypsinis inactivated by adding 1 ml soybean trypsin inhibitor. The cells areplated at 100,000 cells per well in 96 well plates in DMEM/F12supplemented with N₂. Cells for all experiments are grown at 37° C. in awater saturated atmosphere of 5% CO₂. After 2–3 days in culture, cellsare fixed using 4% paraformaldehyde, and then stained using CellTrackerGreen CMFDA. Rho 4D2 (ascites or IgG 1:100), a monoclonal antibodydirected towards the visual pigment rhodopsin is used to detect rodphotoreceptor cells by indirect immunofluorescence. The results arecalculated as % survival: total number of calcein—rhodopsin positivecells at 2–3 days in culture, divided by the total number of rhodopsinpositive cells at time 2–3 days in culture. The total cells(fluorescent) are quantified at 20× objective magnification using a CCDcamera and NIH image software for Macintosh. Fields in the well arechosen at random.

The following polypeptides tested positive in this assay: PRO200,PRO322, PRO540, PRO846 and PRO617.

Example 112 Ability of PRO Polypeptides to Stimulate the Release ofProteoglycans from Cartilage (Assay 97)

The ability of various PRO polypeptides to stimulate the release ofproteoglycans from cartilage tissue was tested as follows.

The metacarphophalangeal joint of 4–6 month old pigs was asepticallydissected, and articular cartilage was removed by free hand slicingbeing careful to avoid the underlying bone. The cartilage was minced andcultured in bulk for 24 hours in a humidified atmosphere of 95% air, 5%CO₂ in serum free (SF) media (DME/F12 1:1) woth 0.1% BSA and 100 U/mlpenicillin and 100 μg/ml streptomycin. After washing three times,approximately 100 mg of articular cartilage was aliquoted into micronicstubes and incubated for an additional 24 hours in the above SF media.PRO polypeptides were then added at 1% either alone or in combinationwith 18 ng/ml interleukin-1α, a known stimulator of proteoglycan releasefrom cartilage tissue. The supernatant was then harvested and assayedfor the amount of proteoglycans using the 1,9-dimethyl-methylene blue(DMB) colorimetric assay (Farndale and Buttle, Biochem. Biophys. Acta883:173–177 (1985)). A positive result in this assay indicates that thetest polypeptide will fmd use, for example, in the treatment ofsports-related joint problems, articular cartilage defects,osteoarthritis or rheumatoid arthritis.

When various PRO polypeptides were tested in the above assay, thepolypeptides demonstrated a marked ability to stimulate release ofproteoglycans from cartilage tissue both basally and after stimulationwith interleulin-1α and at 24 and 72 hours after treatment, therebyindicating that these PRO polypeptides are useful for stimulatingproteoglycan release from cartilage tissue. As such, these PROpolypeptides are useful for the treatment of sports-related jointproblems, articular cartilage defects, osteoarthritis or rheumatoidarthritis. The polypeptides testing positive in this assay are: PRO200.

Example 113 In Vitro Antiproliferative Assay (Assay 161)

The antiproliferative activity of various PRO polypeptides wasdetermined in the investigational, disease-oriented in vitro anti-cancerdrug discovery assay of the National Cancer Institute (NCI), using asulforhodamine B (SRB) dye binding assay essentially as described bySkehan et al., J. Natl. Cancer Inst. 82:1107–1112 (1990). The 60 tumorcell lines employed in this study (“the NCI panel”), as well asconditions for their maintenance and culture in vitro have beendescribed by Monks et al., J. Natl. Cancer Inst. 83:757–766 (1991). Thepurpose of this screen is to initially evaluate the cytotoxic and/orcytostatic activity of the test compounds against different types oftumors (Monks et al., supra Boyd, Cancer: Princ. Pract. Oncol. Update3(10):1–12 [1989]).

Cells from approximately 60 human tumor cell lines were harvested withtrypsin/EDTA (Gibco), washed once, resuspended in IMEM and theirviability was determined. The cell suspensions were added by pipet (100μL volume) into separate 96-well microtiter plates. The cell density forthe 6-day incubation was less than for the 2-day incubation to preventovergrowth. Inoculates were allowed a preincubation period of 24 hoursat 37° C. for stabilization. Dilutions at twice the intended testconcentration were added at time zero in 100 μL aliquots to themicrotiter plate wells (1:2 dilution). Test compounds were evaluated atfive half-log dilutions (1000 to 100,000-fold). Incubations took placefor two days and six days in a 5% CO₂ atmosphere and 100% humidity.

After incubation, the medium was removed and the cells were fixed in 0.1ml of 10% trichloroacetic acid at 40° C. The plates were rinsed fivetimes with deionized water, dried, stained for 30 minutes with 0.1 ml of0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsedfour times with 1% acetic acid to remove unbound dye, dried, and thestain was extracted for five minutes with 0.1 ml of 10 mM Tris base[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) ofsulforhodamine B at 492 nm was measured using a computer-interfaced,96-well microtiter plate reader.

A test sample is considered positive if it shows at least 50% growthinhibitory effect at one or more concentrations. PRO polypeptidestesting positive in this assay are shown in Table 7, where theabbreviations are as follows:

TABLE 7 Test compound Tumor Cell Line Type Cell Line Designation PRO181Leukemia RPMI-8226 PRO181 NSCL NCI-H226; NCI-H522 PRO181 MelanomaMALME-3M; SK-MEL-5 PRO181 Ovarian OVCAR-4 PRO181 Breast NCI/ADR-RESPRO181 Leukemia MOLT-4 PRO181 NSCL NCI-H226* PRO181 CNS SNB-19 PRO181Ovarian OVCAR-3; OVCAR-8 PRO181 Renal A498 PRO181 BreastMDA-MB-231/ATCC; MDA-N PRO181 Melanoma LOX IMVI PRO181 LeukemiaCCRF-CEM; RPMI-8226* PRO181 NSCL HOP-62 PRO181 Leukemia HL-60 (TB)PRO237 Leukemia K-562 PRO237 NSCL NCI-H322M PRO237 Colon HCC-2998;HCT-15 PRO237 Colon KM12 PRO237 Prostate DU-145 PRO237 Breast MDA-NPRO526 NSCL HOP-62; NCI-H322M PRO526 Colon HCT-116 PRO526 Melanoma LOXIMVI; SK-MEL-2 PRO526 Ovarian OVCAR-3 PRO526 Prostate PC-3 PRO526 NSCLNCI-H226 PRO526 CNS SF-539 PRO526 Renal CAKI-1; RXF 393 PRO362 NSCLNCI-H322M PRO362 Colon HCT-116 PRO362 CNS SF-295 PRO362 Melanoma LOXIMVI PRO362 Leukemia MOLT-4; RPMI-8226; SR PRO362 Colon COLO 205 PRO362Breast HS 578T; MDA-N PRO362 Prostate PC-3 PRO362 Leukemia HL-60 (TB);K-562 PRO362 NSCL EKVX; NCI-H23 PRO362 Colon HCC-2998 PRO362 CNS U251PRO362 Melanoma UACC-257; UACC-62 PRO362 Ovarian OVCAR-8 PRO362 BreastT-47D PRO362 NSCL NCI-H522 PRO362 Renal RXF 393; UO-31 PRO362 BreastMDA-MB-435 PRO362 NSCL HOP-62; NCI-H522 PRO362 Colon KM12 PRO362Melanoma MALME-3M; SK-MEL-2 PRO362 Melanoma SK-MEL-28; SK-MEL-5 PRO362Ovarian OVCAR-3; OVCAR-4 PRO362 Breast MCF7 PRO866 Leukemia HL-60 (TB);MOLT-4; SR PRO866 NSCL HOP-62 PRO866 NSCL HOP-92 PRO866 Colon KM12PRO866 CNS SF-295 PRO866 Ovarian IGROV1 PRO866 Breast MDA-MB-435 PRO866Melanoma LOX IMVI PRO320 Leukemia CCRF-CEM; RPMI-8226 PRO320 NSCL HOP62;NCI H322M PRO320 Colon HCT-116 PRO320 Renal SN12C PRO320 Breast MDA-NPRO320 Ovarian OVCAR-3 PRO320 Melanoma MALME-3M *cytotoxic NSCL =non-small cell lung carcinoma CNS = central nervous system

The results of these assays demonstrate that the positive testing PROpolypeptides are useful for inhibiting neoplastic growth in a number ofdifferent tumor cell types and may be used therapeutically therefor.Antibodies against these PRO polypeptides are useful for affinitypurification of these useful polypeptides. Nucleic acids encoding thesePRO polypeptides are useful for the recombinant preparation of thesepolypeptides.

Example 114 Gene Amplification in Tumors

This example shows that certain PRO polypeptide-encoding genes areamplified in the genome of certain human lung, colon and/or breastcancers and/or cell lines. Amplification is associated withoverexpression of the gene product, indicating that the polypeptides areuseftu targets for therapeutic intervention in certain cancers such ascolon, lung, breast and other cancers and diagnostic determination ofthe presence of those cancers. Therapeutic agents may take the form ofantagonists of the PRO polypeptide, for example, murine-human chimeric,humanized or human antibodies against a PRO polypeptide.

The starting material for the screen was genomic DNA isolated from avariety cancers. The DNA is quantitated precisely, e.g.,fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TaqMan™) and real-time quantitative PCR(for example, ABI Prizm7700 Sequence Detection System™ (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding the PRO polypeptide isover-represented in any of the primary lung or colon cancers or cancercell lines or breast cancer cell lines that were screened. The primarylung cancers were obtained from individuals with tumors of the type andstage as indicated in Table 8. An explanation of the abbreviations usedfor the designation of the primary tumors listed in Table 8 and theprimary tumors and cell lines referred to throughout this example aregiven below.

The results of the TaqMan™ are reported in delta (Δ) Ct units. One unitcorresponds to 1 PCR cycle or approximately a 2-fold amplificationrelative to normal, two units corresponds to 4-fold, 3 units to 8-foldamplification and so on. Quantitation was obtained using primers and aTaqMan™ fluorescent probe derived from the PRO polypeptide-encodinggene. Regions of the PRO polypeptide-encoding gene which are most likelyto contain unique nucleic acid sequences and which are least likely tohave spliced out introns are preferred for the primer and probederivation, e.g., 3′-untranslated regions. The sequences for the primersand probes (forward, reverse and probe) used for the PRO polypeptidegene amplification analysis were as follows:

PRO853 (DNA48227-1350) 48227.tm.f1 5′-GGCACTTCATGGTCCTTGAAA-3′ (SEQ IDNO:539) 48227.tm.p1 5′-GGATGTGTGTGAGGCCATGCC-3′ (SEQ ID NO:540)48227.tm.r1 5′-GAAAGTAACCACGGAGGTCAAGAT-3′ (SEQ ID NO:541) PRO1017(DNA56112-1379): 56112.tm.f1 5′-CCTCCTCCGAGACTGAAAGCT-3′ (SEQ ID NO:542)56112.tm.p1 5′-TCGCGTTGCTTTTTCTCGCGTG-3′ (SEQ ID NO:543) 56112.tm.r15′-GCGTGCGTCAGGTTCCA-3′ (SEQ ID NO:544) PRO213-1 (DNA30943-1163-1):30943.tm.f3: 5′-CGTTCGTGCAGCGTGTGTA-3′ (SEQ ID NO:545) 30943.tm.p3:5′-CTTCCTCACCACCTGCGACGGG-3′ (SEQ ID NO:546) 30943.tm.r3:5′-GGTAGGCGGTCCTATAGATGGTT-3′ (SEQ ID NO:547) 30943.tm.f1:5′-AGATGTGGATGAATGCAGTGCTA-3′ (SEQ ID NO:548) 30943.tm.p1:5′-ATCAACACCGCCGGCAGTTACTGG-3′ (SEQ ID NO:549) 30943.tm.r1:5′-ACAGAGTGTACCGTCTGCAGACA-3′ (SEQ ID NO:550) 30943.3trn-5:5′-AGCCTCCTGGTGCACTCCT-3′ (SEQ ID NO:551) 30943.3trn-probe:5′-CGACTCCCTGAGCGAGCAGATTTCC-3′ (SEQ ID NO:552) 30943.3trn-3:5′-CTGGGCAGTCACGAGTCTT-3′ (SEQ ID NO:553) PRO237 (DNA34353-1428):34353.tm.f: 5′-AATCCTCCATCTCAGATCTTCCAG-3′ (SEQ ID NO:554) 34353.tm.p:5′-CCTCAGCGGTAACAGCCGGCC-3′ (SEQ ID NQ:555) 34353.tm.r:5′-TGGGCCAAGGGCTGC-3′ (SEQ ID NO:556) PRO324 (DNA36343-1310):36343.tmf1: 5′-TGGTGGATAACCAACAAGATGG-3′ (SEQ ID NO:557) 36343.tmp1:5′-GAGTCTGCATCCACACCACTCTTAAAGTTCTCAA-3′ (SEQ ID NO:558) 36343.tmr1:5′-AGGTGCTCTTTTCAGTCATGTTT-3′ (SEQ ID NO:559) PRO351 (DNA40571-1315):40571.tm.f1: 5′-TGGCCATTCTCAGGACAAGAG-3′ (SEQ ID NO:560) 40571.tm.p1:5′-CAGTAATGCCATTTGCCTGCCTGCAT-3′ (SEQ ID NO:561) 40571.tm.r1:5′-TGCCTGGAATCACATGACA-3′ (SEQ ID NO:562) PRO362 (DNA45416-1251):45416.tm.f1: 5′-TGTGGCACAGACCCAATCCT-3′ (SEQ ID NO:563) 45416.tm.p1:5′-GACCCTGAAGGCCTCCGGCCT-3′ (SEQ ID NO:564) 45416.tm.r1:5′-GAGAGAGGGAAGGCAGCTATGTC-3′ (SEQ ID NO:565) PRO615 (DNA48304-1323):48304.tm.f1: 5′-CAGCCCCTCTCTTTCACCTGT-3′ (SEQ ID NO:566) 48304.tm.p1:5′-CCATCCTGTGCAGCTGACACACAGC-3′ (SEQ ID NO:567) 48304.tm.r1:5′-GC CAGGCTATGA GGCTCCTT -3′ (SEQ ID NO:568) PRO531 (DNA48314-1320):48314.tm.f1: 5′-TTCAAGTTCCTGAAGCCGATTAT-3′ (SEQ ID NO:569) 48814.tm.p1:5′-CAACTTCCCTCCCCAGTGCCCT-3′ (SEQ ID NO:570) 48814.tm.r1:5′-TTGGGGAAGGTAGAATTTCCTTGTAT-3′ (SEQ ID NO:571) PRO618 (DNA49152-1324):49152.tm.f1: 5′-CCCTTCTGCCTCCCAATTCT-3′ (SEQ ID NO:572) 49152.tm.p1:5′-TCTCCTCCGTCCCCTTCCTCCACT -3′ (SEQ ID NO:573) 49152.tm.r1:5′-TGAGCCACTGCCTTGCATTA-3′ (SEQ ID NO:574) PRO772 (DNA49645-1347):49645.tm.f2: 5′-TCTGCAGACGCGATGGATAA-3′ (SEQ ID NO:575) 49645.tm.p2:5′-CCGAAAATAAAACATCGCCCCTTCTGC-3′ (SEQ ID NO:576) 49645.tm.r2:5′-CACGTGGCCTTTCACACTGA-3′ (SEQ ID NO:577) 49645.tm.f1:5′-ACTTGTGACAGCAGTATGCTGTCTT-3′ (SEQ ID NO:578) 49645.tm.p1:5′-AAGCTTCTGTTCAATCCCAGCGGTCC-3′ (SEQ ID NO:579) 49645.tm.r1:5′-ATGCACAGGCTTTTTCTGGTAA-3′ (SEQ ID NO:580) PRO703 (DNA50913-1287):50913.tm.f1: 5′-GCAGGAAACCTTCGAATCTGAG-3′ (SEQ ID NO:581) 50913.tm.p1:5′-ACACCTGAGGCACCTGAGAGAGGAACTCT-3′ (SEQ ID NO:582) 50913.tm.r1:5′-GACAGCCCAGTACACCTGCAA-3′ (SEQ ID NO:583) PRO792 (DNA56352-1358):56352.tm.f1: 5′-GACGGCTGGATCTGTGAGAAA-3′ (SEQ ID NO:584) 56352.tm.p1:5′-CACAACTGCTGACCCCGCCCA-3′ (SEQ ID NO:585) 56352.tm.r1:5′-CCAGGATACGACATGCTGCAA-3′ (SEQ ID NO:586) PRO474 (DNA56045-1380):56045.tm.f1: 5′-AAACTCCAACCTGTATCAGATGCA-3′ (SEQ ID NO:587) 56045.tm.p1:5′-CCCCCAAGCCCTTAGACTCTAAGCCC-3′ (SEQ ID NO:588) 56045.tm.r1:5′-GACCCGGCACCTTGCTAAC-3′ (SEQ ID NO:589) PRO274 (DNA39987-1184):39987.tm.f: 5′-GGACGGTCAGTCAGGATGACA-3′ (SEQ ID NO:590) 39987.tm.p:5′-TTCGGCATCATCTCTTCCCTCTCCC-3′ (SEQ ID NO:591) 39987.tm.r:5′-ACAAAAAAAAGGGAACAAAATACGA-3′ (SEQ ID NO:592) PRO381 (DNA44194-1317)44194.tm.f: 5′-CTTTGAATAGAAGACTTCTGGACAATTT-3′ (SEQ ID NO:593)44194.tm.p: 5′-TGCAACTGGGAATATACCACGACATGAGA-3′ (SEQ ID NO:594)44194.tm.r: 5′-TAGGGTGCTAATTTGTGCTATAACCT-3′ (SEQ ID NO:595)44194.tm.f2: 5′-GGCTCTGAGTCTCTGCTTGA-3′ (SEQ ID NO:596) 44194.tm.p2:5′-TCCAACAACCATTTTCCTCTGGTCC-3′ (SEQ ID NO:597) 44194.tm.r2:5′-AAGCAGTAGCCATTAACAAGTCA-3′ (SEQ ID NO:598) PRO717 (DNA50988-1326):50988.tm.f3: 5′-CAAGCGTCCAGGTTTATTGA-3′ (SEQ ID NO:599) 50988.tm.r3:5′-GACTACAAGGCGCTCAGCTA-3′ (SEQ ID NO:600) 50988.tm.p3:5′-CCGGCTGGGTCTCACTCCTCC-3′ (SEQ ID NO:601) PRO1330 and PRO1449(DNA64907-1163 and DNA64908-1163. respectively): 30943.tm.f3:5′-CGTTCGTGCAGCGTGTGTA-3′ (SEQ ID NO:602) 30943.tm.p3:5′-CTTCCTCACCACCTGCGACG GG-3′ (SEQ ID NO:603) 30943.tm.r3:5′-GGTAGGCGGTCCTATAGATGGT-3′ (SEQ ID NO:604) 30943.tm.f1:5′-AGATG TGGATGAATG CAGTGCTA-3′ (SEQ ID NO:605) 30943.tm.p1:5′-ATCAACACCGCCGGCAGTTACTGG-3′ (SEQ ID NO:606) 30943.tm.r1:5′-ACAGAGTGTACCGTCTGCAGACA-3′ (SEQ ID NO:607) 30943.3trn-5:5′-AGCCTCCTGGTGCACTCCT-3′ (SEQ ID NO:608) 30943.3trn-probe:5′-CGACTCCCTGAGCGAGCAGATTTCC-3′ (SEQ ID NO:609) 30943.3trn-3:5′-GCTGGGCAGTCACGAGTCTT-3′ (SEQ ID NO:610)

The 5′ nuclease assay reaction is a fluorescent PCR-based techniquewhich makes use of the 5′ exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers (forward [.f] and reverse [.r]) are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe (.p), isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI Prism 7700TM Sequence Detection. The system consists ofa thermocycler, laser, charge-coupled device (CCD) camera and computer.The system amplifies samples in a 96-well format on a thermocycler.During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe CCD. The system includes software for running the instrument and foranalyzing the data.

5′ Nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ΔCt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing cancer DNA results to normal human DNA results.

Table 8 describes the stage T stage and N stage of various primarytumors which were used to screen the PRO polypeptide compounds of theinvention.

TABLE 8 Primary Lung and Colon Tumor Profiles Primary Tumor Stage StageOther Stage Dukes Stage T Stage N Stage Human lung tumor AdenoCa(SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa (SRCC725) [LT1a] IIB T3N0 Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 N0 Human lung tumorAdenoCa (SRCC727) [LT3] IIIA T1 N2 Human lung tumor AdenoCa (SRCC728)[LT4] IB T2 N0 Human lung tumor SqCCa (SRCC729) [LT6] IB T2 N0 Humanlung tumor Aden/SqCCa (SRCC730) [LT7] IA T1 N0 Human lung tumor AdenoCa(SRCC731) [LT9] IB T2 N0 Human lung tumor SqCCa (SRCC732) [LT10] IIB T2N1 Human lung tumor SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumorAdenoCa (SRCC734) [LT12] IV T2 N0 Human lung tumor AdenoSqCCa (SRCC735)[LT13] IB T2 N0 Human lung tumor SqCCa (SRCC736) [LT15] IB T2 N0 Humanlung tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumor SqCCa(SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18] IB T2N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human lung tumorLCCa (SRCC741) [LT21] IIB T3 N1 Human lung AdenoCa (SRCC811) [LT22] 1AT1 N0 Human colon AdenoCa (SRCC742) [CT2] M1 D pT4 N0 Human colonAdenoCa (SRCC743) [CT3] B pT3 N0 Human colon AdenoCa (SRCC744) [CT8] BT3 N0 Human colon AdenoCa (SRCC745) [CT10] A pT2 N0 Human colon AdenoCa(SRCC746) [CT12] MO, R1 B T3 N0 Human colon AdenoCa (SRCC747) [CT14]pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC748) [CT15] M1, R2 D T4 N2Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pN0 Human colon AdenoCa(SRCC750) [CT17] C1 pT3 pN1 Human colon AdenoCa (SRCC751) [CT1] MO, R1 BpT3 N0 Human colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human colon AdenoCa(SRCC753) [CT5] G2 C1 pT3 pN0 Human colon AdenoCa (SRCC754) [CT6] pMO,RO B pT3 pN0 Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0 Humancolon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon AdenoCa (SRCC757)[CT11] B T3 N0 Human colon AdenoCa (SRCC758) [CT18] MO, RO B pT3 pN0DNA Preparation:

DNA was prepared from cultured cell lines, primary tumors, normal humanblood. The isolation was performed using purification kit, buffer setand protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

Cell Culture Lysis:

Cells were washed and trypsinized at a concentration of 7.5×10⁸ per tipand pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation. Thepellets were washed a third time, the suspended cells collected andwashed 2× with PBS. The cells were then suspended into 10 ml PBS. BufferC1 was equilibrated at 4° C. Qiagen protease #19155 was diluted into6.25 ml cold ddH₂O to a final concentration of 20 mg/ml and equilibratedat 4° C. 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse Astock (100 mg/ml) to a final concentration of 200 μg/ml.

Buffer C1 (10 ml, 4° C.) and ddH20 (40 ml, 4° C.) were then added to the10 ml of cell s by inverting and incubated on ice for 10 minutes. Thecell nuclei were pelleted by centrifuging in a Beckman swinging bucketrotor at 2500 rpm at 4° C. for 15 minutes. The supernatant was discardedand the nuclei were suspended with a vortex into 2 ml Buffer C1 (at 4°C.) and 6 ml ddH₂O, followed by a second 4° C. centrifugation at 2500rpm for 15 minutes. The nuclei were then resuspended into the residualbuffer using 200 μl per tip. G2 buffer (10 ml) was added to thesuspended nuclei while gentle vortexing was applied. Upon completion ofbuffer addition, vigorous vortexing was applied for 30 seconds. Quiagenprotease (200 μl, prepared as indicated above) was added and incubatedat 50° C. for 60 minutes. The incubation and centrifugation was repeateduntil the lysates were clear (e.g., incubating additional 30–60 minutes,pelleting at 3000×g for 10 min., 4° C.).

Solid Human Tumor Sample Preparation and Lysis:

Tumor samples were weighed and placed mto 50 ml conical tubes and heldon ice. Processing was limited to no more than 250 mg tissue perpreparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at2×30 seconds each in 2 LddH₂O, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30–60 minutes, pelleting at 3000×g for 10min., 4° C.).

Human Blood Preparation and Lysis:

Blood was drawn from healthy volunteers using standard infectious agentprotocols and citrated into 10 ml samples per tip. Quiagen protease wasfreshly prepared by dilution into 6.25 ml cold ddH₂O to a finalconcentration of 20 mg/ml and stored at 4° C. G2 buffer was prepared bydiluting RNAse A to a final concentration of 200 μg/ml from 100 mg/mlstock. The blood (10 ml) was placed into a 50 ml conical tube and 10 mlC1 buffer and 30 ml ddH₂O (both previously equilibrated to 4° C.) wereadded, and the components mixed by inverting and held on ice for 10minutes. The nuclei were pelleted with a Beckman swinging bucket rotorat 2500 rpm, 4° C. for 15 minutes and the supernatant discarded. With avortex, the nuclei were suspended into 2 ml C1 buffer (4° C.) and 6 mlddH₂O (4° C.). Vortexing was repeated until the pellet was white. Thenuclei were then suspended into the residual buffer using a 200 μl tip.G2 buffer (10 ml) were added to the suspended nuclei while gentlyvortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (2001) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30–60 minutes, pelleting at 3000×g for 10min., 4° C.).

Purification of Cleared Lysates:

(1) Isolation of Genomic DNA:

Genomic DNA was equilibrated (1 sample per maxi tip preparation) with 10ml QBT buffer. QF elution buffer was equilibrated at 50° C. The sampleswere vortexed for 30 seconds, then loaded onto equilibrated tips anddrained by gravity. The tips were washed with 2×15 ml QC buffer. The DNAwas eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15 mlQF buffer (50° C.). Isopropanol (10.5 ml) was added to each sample, thetubes covered with parafin and mixed by repeated inversion until the DNAprecipitated. Samples were pelleted by centrifugation in the SS-34 rotorat 15,000 rpm for 10 minutes at 4° C. The pellet location was marked,the supernatant discarded, and 10 ml 70% ethanol (4° C.) was added.Samples were pelleted again by centrifugation on the SS-34 rotor at10,000 rpm for 10 minutes at 49° C. The pellet location was marked andthe supernatant discarded. The tubes were then placed on their side in adrying rack and dried 10 minutes at 37° C., taking care not to overdrythe samples.

After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) andplaced at 50° C. for 1–2 hours. Samples were held overnight at 4° C. asdissolution continued. The DNA solution was then transferred to 1.5 mltubes with a 26 gauge needle on a tuberculin syringe. The transfer wasrepeated 5× in order to shear the DNA. Samples were then placed at 50°C. for 1–2 hours.

(2) Quantitation of Genomic DNA and Preparation for Gene AmplificationAssay:

The DNA levels in each tube were quantified by standard A₂₆₀, A₂₈₀spectrophotometry on a 1:20 dilution (5 μl DNA+95 μl ddH₂O) using the0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer. A₂₆₀/A₂₈₀ratios were in the range of 1.8–1.9. Each DNA samples was then dilutedfurther to approximately 200 ng/ml in TE (pH 8.5). If the originalmaterial was highly concentrated (about 700 ng/μl), the material wasplaced at 50° C. for several hours until resuspended.

Fluorometric DNA quantitation was then performed on the diluted material(20–600 ng/ml) using the manufacturer's guidelines as modified below.This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes. The Hoechst dye working solution(#H33258, 10 μl, prepared within 12 hours of use) was diluted into 100ml 1×TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400+/−10 units. Each sample wasthen read at least in triplicate. When 3 samples were found to be within10% of each other, their average was taken and this value was used asthe quantification value.

The fluorometricly determined concentration was then used to dilute eachsample to 10 ng/μl in ddH₂O. This was done simultaneously on alltemplate samples for a single TaqMan plate assay, and with enoughmaterial to run 500–1000 assays. The samples were tested in triplicatewith Taqman™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 Ct. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8–9 plates or 64 tests.

Gene Amplification Assay:

The PRO polypeptide compounds of the invention were screened in thefollowing primary tumors and the resulting ΔCt values greater than orequal to 1.0 are reported in Table 9 below.

TABLE 9 ΔCt values in lung and colon primary tumor and cell line modelsPRO1330 Tumor or PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PROPRO PRO PRO and Cell Line 213-1 237 324 351 362 615 531 853 1017 618 772703 792 474 274 381 717 PRO1449 LT-1 1.60 — — — — — — — — — — — — — — —— 1.60 LT-1a 1.24 1.04 — — — — 1.70 — 1.785 — 1.33 1.22 1.16  1.94 — — —1.24 1.62 LT2 — — — — 1.39 — — — — — — — — — — — — — LT3 1.51 1.74 — — —1.31 1.95 — 2.38  1.03 1.11 1.77 1.10  2.55 — — — 1.51 1.55 1.24 1.521.44 LT4 2.26 — — — 1.00 — 1.46 — — — — — — — 1.24 — — 2.26 LT6 1.561.16 — — — 1.00 2.07 — 2.80  — 1.07 1.15 1.81  2.10 — — — 1.56 2.28 LT72.45 1.44 — — — 1.09 — — 1.12  — — 1.44 — 1.06 — — — 2.45 1.03 LT9 1.24— — 1.19 — 1.04 1.10 — 2.74  1.39 1.62 — 1.99  2.56 — — — 1.24 1.14 1.112.59 LT10 — 1.20 — 1.06 1.69 1.18 1.96 — 3.52  1.29 1.46 1.48 2.00  2.63— — — — 1.11 1.16 1.29 2.85 LT11 2.26 — 1.34 1.02 — 1.46 1.79 1.03 1.54 1.84 1.45 1.90 1.20  1.36 — — — 2.26 2.85 1.72 2.94  1.83 5.21 2.85 2.251.27 1.41  2.25 1.79 1.25 1.79 1.06 LT12 1.86 — 1.92 — — 2.08 1.86 1.181.77  — — 1.38 — 1.64 — — — 1.86 4.32 1.87 3.02  1.62 5.01 4.32 2.591.41 1.82  2.59 1.55 1.50 1.55 1.25 LT13 1.98 1.05 — 1.23 — 1.39 2.531.33 1.55  — 1.18 1.33 1.33  1.03 — — 7.03 1.98 2.52 1.09 2.06 2.14 1.20 1.00 2.52 2.38 1.03 1.31 2.03  4.54 2.38 1.14 1.65 LT15 1.40 — —1.14 — 1.67 2.56 1.28 2.23  — 1.47 1.45 1.04  1.35 — — 2.71 1.40 1.581.47 2.95 2.01  1.44 1.86 1.58 2.69 1.09 1.31 2.50  4.97 2.69 1.05 1.52LT16 1.22 1.22 1.63 1.09 — 1.32 — 1.33 2.98  — — 1.07 — 4.23 1.00 — 5.481.22 2.77 1.38 1.77  1.52 2.77 1.75 1.17 1.75 LT17 4.58 1.07 1.75 1.46 —1.66 1.12 1.21 2.90  1.04 1.42 1.24 1.35  1.40 — — — 4.58 3.73 1.59 1.531.62  1.61 1.115 5.45 3.73 5.55 1.21 5.55 1.50 1.13 LT18 — — — 1.07 — —— — 3.28  — — — — 5.31 1.61 — — — 1.68  LT19 1.03 — 1.90 1.33 — 1.592.08 — 2.54  — 1.60 1.38 1.62  1.59 — — — 1.03 1.22 1.50 2.95 2.98  1.191.84 1.22 1.26 1.03 1.21  4.84 1.26 1.48 LT21 1.86 — 1.15 1.27 — 1.19 —— 3.14  — — 1.22 — 5.15 — — — 1.86 1.83 1.09 1.83 3.21 1.06 3.21 LT221.61 — — — — — — — — — — — — — — — — 1.61 CT2 1.61 — — — — 1.36 2.21 2.43.72  — — 2.10 1.46  2.67 — — — 1.61 2.11 1.25 2.55 2.55  1.65 2.11 1.901.48 CT3 — — — — — 1.12 1.50 1.52 3.91  — — 1.62 — 2.41 — — — — 1.58 1.02 CT8 2.80 — — — — — 1.15 1.55 2.66  — — 1.06 — 2.34 — — — 2.80 1.34CT10 2.39 — — — — 1.55 1.75 1.97 3.57  — — 1.96 — 2.23 — — — 2.39 1.471.78  1.21 CT12 3.45 — — — — 1.08 1.93 1.36 3.50  — — 1.57 — 2.46 — — —3.45 1.30 1.08  CT14 3.79 — — — — 1.76 1.47 1.75 3.88  — — 1.19 — 2.83 —— — 3.79 1.02 1.11 1.86  CT15 3.66 — — — — 1.23 2.44 1.75 3.62  — — 1.70— 2.89 — — 2.61 3.66 1.33 CT16 2.66 — — — — 1.29 1.95 1.11 3.12  — —1.51 — 2.60 — — 2.21 2.66 CT17 3.63 — — — — 1.44 2.19 1.11 3.34  — —1.31 — 2.33 — — 3.31 3.63 CT1 — — — — — — — 1.09 — — — 1.08 — 1.00 — — —— CT4 1.18 — — — — 1.17 — 1.16 1.11  — — 1.63 — 1.13 — — — 1.18 1.07 CT51.25 — — — — 1.12 1.59 1.95 2.21  — — 1.50 — 1.84 — — — 1.25 1.16 1.352.05 2.11 CT6 1.27 — — — — — — — 1.12  — — 1.38 — 1.24 — — — 1.27 1.36CT7 — — — — — — — 1.14 — — — 1.50 — — — — — — CT9 — — — — — — 1.28 —1.29  — — — — — — — — — CT11 — — — — — 1.74 1.49 1.88 1.48  — — 1.99 —2.11 — — — — 1.17 2.13 CT18 — — — — — 1.36 — — — — — 1.15 — 9.66 — — — —Calu-1 1.35 — — — — — — — — — — — — — — — 1.77 1.35 2.95 2.95 H441 2.00— — — — — — 1.71 — — — — — — — — 2.57 2.00 H522 — — — — — — — 1.03 — — —— — — — — 3.78 — H810 2.76 — — — — — — — — — — — — — — — 1.84 2.76 HT291.31 — — — — — — — — — — — — — — — 1.71 1.31 SW403 2.08 — — — — — — — —— — — — — — — 2.09 2.08 LS174T 1.61 — — — — — — — — — — — — — — — 2.901.61 HCT15 1.22 — — — — — — — — — — — — — — — 1.46 1.22 HCC2998 1.73 — —— — — — — — — — — — — — — 1.20 1.73 HF-00643 — — — — — — — — — — — — — —— 4.83 — — HF-000840 — — — — — — — — — — — — — — — 1.08 — — HF-000811 —— — — — — — — — — — — — — — 2.09 — — 3.15 HF-001294 — — — — — — — — — —— — — — — 1.14 — — 1.08 HF-001296 — — — — — — — — — — — — — — — 3.18 — —3.53 HF-001291 — — — — — — — — — — — — — — — 1.17 — — A549 — — — — — — —— — — — — — — — — 1.66 — H460 — — — — — — — — — — — — — — — — 2.50 —SKMES1 — — — — — — — — — — — — — — — — 2.15 — SW620 — — — — — — — — — —— — — — — — 2.36 — Colo320 — — — — — — — — — — — — — — — — 1.99 — 2.73HCT116 — — — — — — — — — — — — — — — — 1.90 — SKCO1 — — — — — — — — — —— — — — — — 3.13 — Colo205 — — — — — — — — — — — — — — — — 1.48 — KM12 —— — — — — — — — — — — — — — — 1.67 —Summary

Because amplification of the various DNA's as described above occurs invarious tumors, it is likely associated with tumor formation and/orgrowth. As result, antagonists (e.g., antibodies) directed against thesepolypeptides would be expected to be useful in cancer therapy.

Example 115 Induction of c-fos in Endothelial Cells (Assay 34)

This assay is designed to determine whether PRO polypeptides show theability to induce c-fos in endothelial cells. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) ingrowth media (50% Ham's P12 w/o GHT: low glucose, and 50% DMEM withoutglycine: with NaHCO3, 1% glutamine, 10 mM HEPES, 10% PBS, 10 ng/ml bFGF)were plated on 96-well microtiter plates at a cell density of 1×10⁴cells/well. The day after plating, the cells were starved by removingthe growth media and treating the cells with 100 μl/well test samplesand controls (positive control=growth media; negative control=Protein32buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol pH 6.8). The cellswere incubated for 30 minutes at 37° C., in 5% CO₂. The samples wereremoved, and the first part of the bDNA kit protocol (ChironDiagnostics, cat. #6005-037) was followed, where each capitalizedreagent/buffer listed below was available from the kit.

Briefly, the amounts of the TM Lysis Buffer and Probes needed for thetests were calculated based on information provided by the manufacturer.The appropriate amounts of thawed Probes were added to the TM LysisBuffer. The Capture Hybridization Buffer was warmed to room temperature.The bDNA strips were set up in the metal strip holders, and 100 μl ofCapture Hybridization Buffer was added to each b-DNA well needed,followed by incubation for at least 30 minutes. The test plates with thecells were removed from the incubator, and the media was gently removedusing the vacuum manifold. 100 μl of Lysis Hybridization Buffer withProbes were quickly pipetted into each well of the microtiter plates.The plates were then incubated at 55° C. for 15 minutes. Upon removalfrom the incubator the plates were placed on the vortex mixer with themicrotiter adapter head and vortexed on the #2 setting for one minute.80 μl of the lysate was removed and added to the bDNA wells containingthe Capture Hybridization Buffer, and pipetted up and down to mix. Theplates were incubated at 53° C. for at least 16 hours.

On the next day, the second part of the bDNA kit protocol was followed.Specifically, the plates were removed from the incubator and placed onthe bench to cool for 10 minutes. The volumes of additions needed werecalculated based upon information provided by the manufacturer. AnAmplifier Working Solution was prepared by making a 1:100 dilution ofthe Amplifier Concentrate (20 fm/μl) in AL Hybridization Buffer. Thehybridization mixture was removed from the plates and washed twice withWash A. 50 μl of Amplifier Working Solution was added to each well andthe wells were incubated at 53° C. for 30 minutes. The plates were thenremoved from the incubator and allowed to cool for 10 minutes. The LabelProbe Working Solution was prepared by making a 1:100 dilution of LabelConcentrate (40 pmoles/μl) in AL Hybridization Buffer. After the10-minute cool-down period, the amplifier hybridization mixture wasremoved and the plates were washed twice with Wash A. 50 μl of LabelProbe Working Solution was added to each well and the wells wereincubated at 53° C. for 15 minutes. After cooling for 10 minutes, theSubstrate was warmed to room temperature. Upon addition of 3 μl ofSubstrate Enhancer to each ml of Substrate needed for the assay, theplates were allowed to cool for 10 minutes, the label hybridizationmixture was removed, and the plates were washed twice with Wash A andthree times with Wash D. 50 μl of the Substrate Solution with Enhancerwas added to each well. The plates were incubated for 30 minutes at 37°C. and RLU was read in an appropriate luminometer.

The replicates were averaged and the coefficient of variation wasdetermined. The measure of activity of the fold increase over thenegative control (Protein 32/HEPES buffer described above) value wasindicated by chemiluminescence units (RLU). The results are consideredpositive if the PRO polypeptide exhibits at least a two-fold value overthe negative buffer control. Negative control=1.00 RLU at 1.00%dilution. Positive control=8.39 RLU at 1.00% dilution.

The following PRO polypeptides tested positive in this assay: PRO938,PRO200, PRO865, PRO788 and PRO1013.

Example 116 Proliferation of Rat Utricular Supporting Cels (Assay 54)

This assay shows that certain polypeptides of the invention act aspotent mitogens for inner ear supporting cells which are auditory haircell progenitors and, therefore, are useful for inducing theregeneration of auditory hair cells and treating hearing loss inmammals. The assay is performed as follows. Rat UEC4 utricularepithelial cells are aliquoted into 96 well plates with a density of3000 cells/well in 200 μl of serum-containing medium at 33° C. The cellsare cultured overnight and are then switched to serum-free medium at 37°C. Various dilutions of PRO polypeptides (or nothing for a control) arethen added to the cultures and the cells are incubated for 24 hours.After the 24 hour incubation, ³H-thymidine (1 μCi/well) is added and thecells are then cultured for an additional 24 hours. The cultures arethen washed to remove unincorporated radiolabel, the cells harvested andCpm per well determined. Cpm of at least 30% or greater in the PROpolypeptide treated cultures as compared to the control cultures isconsidered a positive in the assay.

The following polypeptide tested positive in this assay: PRO337, PRO363and PRO1012.

Example 117 Detection of PRO Polypeptides that Affect Glucose or FFAUptake by Primary Rat Adipocytes (Assay 94)

This assay is designed to determine whether PRO polypeptides show theability to affect glucose or FFA uptake by adipocyte cells. PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by adipocytes would bebeneficial including, for example, obesity, diabetes or hyper- orhypo-insulinemia.

In a 96 well format, PRO polypeptides to be assayed are added to primaryrat adipocytes, and allowed to incubate overnight. Samples are taken at4 and 16 hours and assayed for glycerol, glucose and FFA uptake. Afterthe 16 hour incubation, insulin is added to the media and allowed toincubate for 4 hours. At this time, a sample is taken and glycerol,glucose and FFA uptake is measured. Media containing insulin without thePRO polypeptide is used as a positive reference control. As the PROpolypeptide being tested may either stimulate or inhibit glucose and FFAuptake, results are scored as positive in the assay if greater than 1.5times or less than 0.5 times the insulin control.

The following PRO polypeptides tested positive as stimulators of glucoseand/or FFA uptake in this assay: PRO181, PRO200, PRO337, PRO362, PRO363,PRO731, PRO534, PRO1114 and PRO1075.

The following PRO polypeptides tested positive as inhibitors of glucoseand/or FFA uptake in this assay: PRO195, PRO322, PRO862, PRO868, PRO865and PRO162.

Example 118 Detection of Polypeptides that Affect Glucose and/or FFAUptake in Skeletal Muscle (Assay 106)

This assay is designed to determine whether PRO polypeptides show theability to affect glucose or FFA uptake by skeletal muscle cells. PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by skeletal muscle would bebeneficial including, for example diabetes or hyper- orhypo-insulinemia.

In a 96 well format, PRO polypeptides to be assayed are added to primaryrat differentiated skeletal muscle, and allowed to incubate overnight.Then fresh media with the PRO polypeptide and +/− insulin are added tothe wells. The sample media is then monitored to determine glucose andFFA uptake by the skeletal muscle cells. The insulin will stimulateglucose and FFA uptake by the skeletal muscle, and insulin in mediawithout the PRO polypeptide is used as a positive control, and a limitfor scoring. As the PRO polypeptide being tested may either stimulate orinhibit glucose and FFA uptake, results are scored as positive in theassay if greater than 1.5 times or less than 0.5 times the insulincontrol.

The following PRO polypeptides tested positive as either stimulatorrs orinhibitors of glucose and/or FFA uptake in this assay: PRO181, PRO200,PRO1083, PRO865, PRO162, PRO1008 and PRO1330.

Example 119 Stimulation of Heart Neonatal Hypertrophy (Assay 1)

This assay is designed to measure the ability of PRO polypeptides tostimulate hypertrophy of neonatal heart. PRO polypeptides testingpositive in this assay are expected to be useful for the therapeutictreatment of various cardiac insufficiency disorders.

Cardiac myocytes from 1-day old Harlan Sprague Dawley rats wereobtained. Cells (180 μL at 7.5×10⁴/ml, serum <0.1%, freshly isolated)are added on day 1 to 96-well plates previously coated with DMEM/F12+4%FCS. Test samples containing the test PRO polypeptide or growth mediumonly (hegative control) (20 μl/well) are added directly to the wells onday 1. PGF (20 μl/well) is then added on day 2 at final concentration of10⁻⁶ M. The cells are then stained on day 4 and visually scored on day5, wherein cells showing no increase in size as compared to negativecontrols are scored 0.0, cells showing a small to moderate increase insize as compared to negative controls are scored 1.0 and cells showing alarge increase in size as compared to negative controls are scored 2.0.A positive result in the assay is a score of 1.0 or greater.

The following polypeptides tested positive In this assay: PRO195,PRO200, PRO526 and PRO792.

Example 120 Enhancement of Heart Neonatal Hypertrophy Induced by F2a(Assay 37)

This assay is designed to measure the ability of PRO polypeptides tostimulate hypertrophy of neonatal heart. PRO polypeptides testingpositive in this assay are expected to be useful for the therapeutictreatment of various cardiac insufficiency disorders.

Cardiac myocytes from l-day old Harlan Sprague Dawley rats wereobtained. Cells (180 μl at 7.5×10⁴/ml, serum <0.1%, freshly isolated)are added on day 1 to 96-well plates previously coated with DMEM/F12+4%FCS. Test samples containing the test PRO polypeptide (20 μl/well) areadded directly to the wells on day 1. PGF (20 μl/well) is then added onday 2 at a final concentration of 10⁻⁶ M. The cells are then stained onday 4 and visually scored on day 5. Visual scores are based on cellsize, wherein cells showing no increase in size as compared to negativecontrols are scored 0.0, cells showing a small to moderate increase insize as compared to negative controls are scored 1.0 and cells showing alarge increase in size as compared to negative controls are scored 2.0.A score of 1.0 or greater is considered positive.

No PBS is included, since calcium concentration is critical for assayresponse. Plates are coated with DMEM/F12 plus 4% FCS (200 μl/well).Assay media included: DMEM/F12 (with 2.44 gm bicarbonate), 10 μ/mltransferrin, 1 μg/ml insulin, 1 μg/ml aprotinin, 2 mmol/L glutamine, 100U/ml penicillin G, 100 μg/ml streptomycin. Protein buffer containingmannitol (4%) gave a positive signal (score 3.5) at 1/10 (0.4%) and1/100 (0.04%), but not at 1/1000 (0.004%). Therefore the test samplebuffer containing mannitol is not run.

The following PRO polypeptides tested positive in this assay: PRO195.

Example 121 Guinea Pig Vascular Leak (Assays 32 and 51)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to induce vascular permeability.Polypeptides testing positive in this assay are expected to be usefulfor the therapeutic treatment of conditions which would benefit fromenhanced vascular permeability including, for example, conditions whichmay benefit from enhanced local immune system cell infiltration.

Hairless guinea pigs weighing 350 grams or more were anesthetized withKetamine (75–80 mg/kg) and 5 mg/kg Xylazine intramuscularly. Testsamples containing the PRO polypeptide or a physiological buffer withoutthe test polypeptide are injected into skin on the back of the testanimals with 100 μl per injection site intradermally. There wereapproximately 16–24 injection sites per animal. One ml of Evans blue dye(1% in PBS) is then injected intracardially. Skin vascular permeabilityresponses to the compounds (i.e., blemishes at the injection sites ofinjection) are visually scored by measuring the diameter (in mm) ofblue-colored leaks from the site of injection at 1 and 6 hours postadministration of the test materials. The mm diameter of blueness at thesite of injection is observed and recorded as well as the severity ofthe vascular leakage. Blemishes of at least 5 mm in diameter areconsidered positive for the assay when testing purified proteins, beingindicative of the ability to induce vascular leakage or permeability. Aresponse greater than 7 mm diameter is considered positive forconditioned media samples. Human VEGF at 0.1 μl/100 μl is used as apositive control, inducing a response of 15–23 mm diameter.

The following PRO polypeptides tested positive in this assay: PRO200.

Example 122 Skin Vascular Permeability Assay (Assay 64)

This assay shows that certain polypeptides of the invention stimulate animmune response and induce inflammation by inducing mononuclear cell,eosinophil and PMN infiltration at the site of injection of the animal.Compounds which stimulate an immune response are useful therapeuticallywhere stimulation of an immune response is beneficial. This skinvascular permeability assay is conducted as follows. Hairless guineapigs weighing 350 grams or more are anesthetized with ketamine (75–80mg/Kg) and 5 mg/Kg xylazine intramuscularly (IM). A sample of purifiedpolypeptide of the invention or a conditioned media test sample isinjected intradermally onto the backs of the test animals with 100 μlper injection site. It is possible to have about 10–30, preferably about16–24, injection sites per animal. One μl of Evans blue dye (1% inphysiologic buffered saline) is injected intracardially. Blemishes atthe injection sites are then measured (mm diameter) at 1 hr and 6 hrpost injection. Animals were sacrificed at 6 hrs after injection. Eachskin injection site is biopsied and fixed in formalin. The skins arethen prepared for histopathologic evaluation. Each site is evaluated forinflammatory cell infiltration into the skin. Sites with visibleinflammatory cell inflammation are scored as positive. Inflammatorycells may be neutrophilic, eosinophilic, monocytic or lymphocytic. Atleast a minimal perivascular infiltrate at the injection site is scoredas positive, no infiltrate at the site of injection is scored asnegative.

The following polypeptide tested positive in this assay: PRO200, PRO362and PRO1031.

Example 123 Induction of c-fos in Cortical Neurons (Assay 83)

This assay is designed to determine whether PRO polypeptides show theability to induce c-fos in cortical neurons. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of nervous system disorders and injuries whereneuronal proliferation would be beneficial.

Cortical neurons are dissociated and plated in growth medium at 10,000cells per well in 96 well plates. After aproximately 2 cellulardivisions, the cells are treated for 30 minutes with the PRO polypeptideor nothing (negative control). The cells are then fixed for 5 minuteswith cold methanol and stained with an antibody directed againstphosphorylated CREB. mRNA levels are then calculated usingchemiluminescence. A positive in the assay is any factor that results inat least a 2-fold increase in c-fos message as compared to the negativecontrols.

The following PRO polypeptides tested positive in this assay: PRO200.

Example 124 Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92)

This assay shows that certain polypeptides of the invention act toinduce proliferation of mammalian kidney mesangial cells and, therefore,are useful for treating kidney disorders associated with decreasedmesangial cell function such as Berger disease or other nephropathiesassociated with Schönlein-Henoch purpura, celiac disease, dermatitisherpetiformis or Crohn disease. The assay is performed as follows. Onday one, mouse kidney mesangial cells are plated on a 96 well plate ingrowth media (3:1 mixture of Dulbecco's modified Eagle's medium andHam's F12 medium, 95% fetal bovine serum, 5% supplemented with 14 mMHEPES) and grown overnight. On day 2, PRO polypeptides are diluted at 2concentrations (1% and 0.1%) in serum-free medium and added to thecells. Control samples are serum-free medium alone. On day 4, 20 μl ofthe Cell Titer 96 Aqueous one solution reagent (Progema) was added toeach well and the colormetric reaction was allowed to proceed for 2hours. The absorbance (OD) is then measured at 490 nm. A positive in theassay is anything that gives an absorbance reading which is at least 15%above the control reading.

The following polypeptide tested positive In this assay: PRO200, PRO363,PRO731, PRO534, PRO866 and PRO1031.

Example 125 Pericyte c-Fos Induction (Assay 93)

This assay shows that certain polypeptides of the invention act toinduce the expression of c-fos in pericyte cells and, therefore, areuseful not only as diagnostic markers for particular types ofpericyte-associated tumors but also for giving rise to antagonists whichwould be expected to be useful for the therapeutic treatment ofpericyte-associated tumors. Specifically, on day 1, pericytes arereceived from VEC Technologies and all but 5 ml of media is removed fromflask. On day 2, the pericytes are trypsinized, washed, spun and thenplated onto 96 well plates. On day 7, the media is removed and thepericytes are treated with 100 μl of PRO polypeptide test samples andcontrols (positive control=DME+5% serum+/−PDGF at 500 ng/ml; negativecontrol=protein 32). Replicates are averaged and SD/CV are determined.Fold increase over Protein 32 (buffer control) value indicated bychemiluminescence units (RLU) luminometer reading verses frequency isplotted on a histogram. Two-fold above Protein 32 value is consideredpositive for the assay. ASY Matrix: Growth media low glucose DMEM 32 20%FBS+1× pen strep+1× fungizone. Assay Media=low glucose DMEM+5% FBS.

The following polypeptides tested positive in this assay: PRO200.

Example 126 Chondrocyte Re-differentiation Assay (Assay 110)

This assay shows that certain polypeptides of the invention act toinduce redifferentiation of chondrocytes, therefore, are expected to beuseful for the treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis. The assay is performedas follows. Porcine chondrocytes are isolated by overnight collagenasedigestion of articulary cartilage of metacarpophalangeal joints of 4–6month old female pigs. The isolated cells are then seeded at 25,000cells/cm² in Ham F-12 containing 10% FBS and 4 μg/ml gentamycin. Theculture media is changed every third day and the cells are then seededin 96 well plates at 5,000 cells/well in 100 μl of the same mediawithout serum and 100 μl of the test PRO polypeptide, 5 nM staurosporin(positive control) or medium alone (negative control) is added to give afinal volume of 200 μl/well. After 5 days of incubation at 37° C., apicture of each well is taken and the differentiation state of thechondrocytes is determined. A positive result In the assay occurs whenthe redifferentiation of the chondrocytes is determined to be moresimilar to the positive control than the negative control.

The following polypeptide tested positive in this assay: PRO200, PRO285,PRO337, PRO526, PRO362, PRO363, PRO531, PRO1083, PRO862, PRO733,PRO1017, PRO792, PRO788, PRO1008, PRO1075, PRO725 and PRO1031.

Example 127 Fetal Hemoglobin Induction in an Erythroblastic Cell Line(Assay 107)

This assay is useful for screening PRO polypeptides for the ability toinduce the switch from adult hemoglobin to fetal hemoglobin in anerythroblastic cell line. Molecules testing positive in this assay areexpected to be useful for therapeutically treating various mammalianhemoglobin-associated disorders such as the various thalassemias. Theassay is performed as follows. Erythroblastic cells are plated instandard growth medium at 1000 cells/well in a 96 well format. PROpolypeptides are added to the growth medium at a concentration of 0.2%or 2% and the cells are incubated for 5 days at 37° C. As a positivecontrol, cells are treated with 100 μM hemin and as negative control,the cells are untreated. After 5 days, cell lysates are prepared andanalyzed for the expression of gamma globin (a fetal marker). A positivein the assay is a gamma globin level at least 2-fold above the negativecontrol.

The following polypeptides tested positive in this assay: PRO237,PRO381, PRO362, PRO724, PRO866, PRO1114, PRO725 and PRO1071.

Example 128 Induction of Pancreatic β-Cell Precursor Proliferation(Assay 117)

This assay shows that certain polypeptides of the invention act toinduce an increase in the number of pancreatic β-Cell precursor cellsand, therefore, are useful for treating various insulin deficient statesin mammals, including diabetes mellitus. The assay is performed asfollows. The assay uses a primary culture of mouse fetal pancreaticcells and the primary readout is an alteration in the expression ofmarkers that represent either β-cell precursors or mature β-cells.Marker expression is measured by real time quantitative PCR (RTQ-PCR);wherein the marker being evaluated is a transcription factor calledPdx1.

The pancreata are dissected from E14 embryos (CD1 mice). The pancreataare then digested with collagenase/dispase in F12/DMEM at 37° C. for 40to 60 minutes (collagenase/dispase, 1.37 mg/ml, Boehringer Mannheim,#1097113). The digestion is then neutralized with an equal volume of 5%BSA and the cells are washed once with RPMI1640. At day 1, the cells areseeded into 12-well tissue culture plates (precoated with laminin, 20μg/ml in PBS, Boehringer Mannheim, #124317). Cells from pancreata from1–2 embryos are distributed per well. The culture medium for thisprimary cuture is 14F/1640. At day 2, the media is removed and theattached cells washed with RPMI/1640. Two mls of minimal media are addedin addition to the protein to be tested. At day 4, the media is removedand RNA prepared from the cells and marker expression analyzed by realtime quantitative RT-PCR. A protein is considered to be active in theassay if it increases the expression of the relevant β-cell marker ascompared to untreated controls.

14F/1640 is RPM11640 (Gibco) plus the following:

group A 1:1000

group B 1:1000

recombinant human insulin 10 μg/ml

Aprotinin (50 μg/ml) 1:2000 (Boehringer manhein #981532)

Bovine pituitary extract (BPE) 60 μg/ml

Gentamycin 100 ng/ml

Group A: (in 10 ml PBS)

Transferrin, 100 mg (Sigma T2252)

Epidermal Growth Factor, 100 μg (BRL 100004)

Triiodothyronine, 10 μl of 5×10⁻⁶ M (Sigma T5516)

Ethanolamine, 100 μl of 10⁻¹ M (Sigma E0135)

Phosphoethalamrine, 100 μl of 10⁻¹ M (Sigma P0503)

Selenium, 4 μl of 10⁻¹ M (Aesar #12574)

Group C: (in 10 ml 100% ethanol)

Hydrocortisone, 2 μl of 5×10⁻³ M (Sigma #H0135)

Progesterone, 100 μl of 1×10⁻³ M (Sigma #P6149)

Forskolin, 500 μl of 20 mM (Calbiochem #344270)

Minimal media:

RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/rml), gentamycin(100 ng/ml), aprotinin (50 μg/ml) and BPE (15 μg/ml).

Defined media:

RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml), gentamycin(100 ng/ml) and aprotinin (50 μg/ml).

The following polypeptides tested positive in this assay: PRO237 andPRO731.

Example 129 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR)Assay (Assay 24)

This example shows that certain polypeptides of the invention are activeas a stimulator of the proliferation of stimulated T-lymphocytes.Compounds which stimulate proliferation of lymphocytes are usefiltherapeutically where enhancement of an immune response is beneficial. Atherapeutic agent may take the form of antagonists of the polypeptide ofthe invention, for example, murine-human chimeric, humanized or humanantibodies against the polypeptide.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J E Coligan, A M Kruisbeek, D HMarglies, E M Shevach, W Strober, National Insitutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammnalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads).

The assay is prepared by plating in triplicate wells a mixture of:

100:1 of test sample diluted to 1% or to 0.1%,

50:1 of irradiated stimulator cells, and

50:1 of responder PBMC cells.

100 microliters of cell culture media or 100 microliter of CD4-IgG isused as the control. The wells are then incubated at 37° C., 5% CO₂ for4 days. On day 5, each well is pulsed with tritiated thymidine (1.0mC/well; Amersham). After 6 hours the cells are washed 3 times and thenthe uptake of the label is evaluated.

In another variant of this assay, PBMCs are isolated from the spleens ofBalblc mice and C57B6 mice. The cells are teased from freshly harvestedspleens assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% nonessential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

Positive increases over control are considered positive with increasesof greater than or equal to 180% being preferred. However, any valuegreater than control indicates a stimulatory effect for the testprotein.

The following PRO polypeptides tested positive in this assay: PRO273,PRO526, PRO381, PRO719, PRO866 and PRO1031.

Example 130 Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay(Assay 67)

This example shows that one or more of the polypeptides of the inventionare active as inhibitors of the proliferation of stimulatedT-lymphocytes. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an immune response isbeneficial.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J E Coligan, A M Kruisbeek, D HMarglies, E M Shevach, W Strober, National Insitutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO2) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads).

The assay is prepared by plating in triplicate wells a mixture of:

100:1 of test sample diluted to 1% or to 0.1%,

50:1 of irradiated stimulator cells, and

50:1 of responder PBMC cells.

100 microliters of cell culture media or 100 microliter of CD4-IgG isused as the control. The wells are then incubated at 37° C., 5% CO₂ for4 days. On day 5, each well is pulsed with tritiated thymidine (1.0mC/well; Amersham). After 6 hours the cells are washed 3 times and thenthe uptake of the label is evaluated.

In another variant this assay, PBMCs are isolated from the spleens ofBalb/c mice and C57B6 mice. The cells are teased from freshly harvestedspleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

Any decreases below control is considered to be a positive result for aninhibitory compound, with decreases of less than or equal to 80% beingpreferred. However, any value less than control indicates an inhibitoryeffect for the test protein.

The following polypeptide tested positive in this assay: PRO273, PRO526,PRO381, PRO701l, PRO363, PRO531, PRO1083, PRO865, PRO788 and PRO1114.

Example 131 Fibroblast (BHK-21) Proliferation (Assay 98)

This assay shows that certain PRO polypeptides of the invention act toinduce proliferation of mammalian fibroblast cells in culture and,therefore, function as useful growth factors in mammalian systems. Theassay is performed as follows. BHK-21 fibroblast cells plated instandard growth medium at 2500 cells/well in a total volume of 100 μl.The PRO polypeptide, β-FGF (positive control) or nothing (negativecontrol) are then added to the wells in the presence of 1 μg/ml ofheparin for a total final volume of 200 μl. The cells are then incubatedat 37° C. for 6 to 7 days. After incubation, the media is removed, thecells are washed with PBS and then an acid phosphatase substratereaction mixture (100 μl/well) is added. The cells are then incubated at37° C. for 2 hours. 10 μl per well of 1N NaOH is then added to stop theacid phosphatase reaction. The plates are then read at OD 405 mn. Apositive in the assay is acid phosphatase activity which is at least 50%above the negative control.

The following PRO polypeptide tested positive in this assay: PRO273 andPRO731.

Example 132 Induction of Endothelial Cell Apootosis (ELISA) (Assay 109)

The ability of PRO polypeptides to induce apoptosis in endothelial cellswas tested in human venous umbilical vein endothelial cells (HUVEC, CellSystems) using a 96-well format, in 0% serum media supplemented with 100ng/ml VEGF, 0.1% BSA, 1×penn/strep. A positive result in this assayindicates the usefulness of the polypeptide for therapeutically treatingany of a variety of conditions associated with undesired endothelialcell growth including, for example, the inhibition of tumor growth. The96-well plates used were manufactured by Falcon (No. 3072). Coating of96 well plates were prepared by allowing gelatinization to occur for >30minutes with 100 μl of 0.2% gelatin in PBS solution. The gelatin mix wasaspirated thoroughly before plating HUEC cells at a final concentrationof 2×10⁴ cells/mi in 10% serum containing medium—100 μl volume per well.The cells were grown for 24 hours before adding test samples containingthe PRO polypeptide of interest.

To all wells, 100 μl of 0% serum media (Cell Systems) complemented with100 ng/ml VEGF, 0.1% BSA, 1×penn/strep was added. Test samplescontaining PRO polypeptides were added in triplicate at dilutions of 1%,0.33% and 0.11%. Wells without cells were used as a blank and wells withcells only were used as a negative control. As a positive control, 1:3serial dilutions of 50 μl of a 3× stock of staurosporine were used. Thecells were incubated for 24 to 35 hours prior to ELISA.

ELISA was used to determine levels of apoptosis preparing solutionsaccording to the Boehringer Manual [Boehringer, Cell Death DetectionELISA plus, Cat No. 1 920 685]. Sample preparations: 96 well plates werespun down at 1 krpm for 10 minutes (200 g); the supernatant was removedby fast inversion, placing the plate upside down on a paper towel removeresidual liquid. To each well, 200 μl of 1× Lysis buffer was added andincubation allowed at room temperature for 30 minutes without shaking.The plates were spun down for 10 minutes at 1 krpm, and 20 μl of thelysate (cytoplasmic fraction) was transferred into streptavidin coatedMTP. 80 μl of immunoreagent mix was added to the 20 μl lystate in eachwell. The MTP was covered with adhesive foil and incubated at roomtempearature for 2 hours by placing it on an orbital shaker (200 rpm).After two hours, the supernatant was removed suction and the wellsrinsed three times with 250 μl of 1× incubation buffer per well (removedby suction). Substrate solution was added (100 μl) into each well andincubated on an orbital shaker at room temperature at 250 rpm untilcolor development was sufficient for a photometric analysis (approx.after 10–20 minutes). A 96 well reader was used to read the plates at405 nm, reference wavelength, 492 nm. The levels obtained for PIN 32(control buffer) was set to 100%. Samples with levels >130% wereconsidered positive for induction of apoptosis.

The following PRO polypeptides tested positive in this assay: PRO846.

Example 133 Induction of Endothelial Cell Apoptosis (Assay 73)

The ability of PRO polypeptides induce apoptosis in endothelial cellswas tested in human venous umbilical vein endothelial cells (HUVEC, CellSystems). A positive test in the assay is indicative of the usefulnessof the polypeptide in therapeutically treating tumors as well asvascular disorders where inducing apoptosis of endothelial cells wouldbe beneficial.

The cells were plated on 96-well microtiter, plates (Amersham LifeScience, cytostar-T scintillating microplate, RPNQ160, sterile,tissue-culture treated individually wrapped), in 10% serum (CSG-medium,Cell Systems), at a density of 2×10⁴ cells per well in a total volume of100 μl. On day 2, test samples containing the PRO polypeptide were addedin triplicate at dilutions of 1%, 0.33% and 0.11%. Wells without cellswere used as a blank and wells with cells only were used as a negativecontrol. As a positive control 1:3 serial dilutions of 50 μl of a 3×stock of staurosporine were used. The ability of the PRO polypeptide toinduce apoptosis was determined by processing of the 96 well plates fordetection of Annexin V, a member of the calcium and phospholipid bindingproteins, to detect apoptosis.

0.2 ml Annexin V—Biotin stock solution (100 μg/ml) was diluted in 4.6 ml2 × Ca²⁺ binding buffer and 2.5% BSA (1:25 dilution). 50 μl of thediluted Annexin V—Biotin solution was added to each well (exceptcontrols) to a final concentration of 1.0 μg/ml. The samples wereincubated for 10–15 minutes with Annexin-Biotin prior to direct additionof ³⁵S-Streptavidin. ³⁵S-Streptavidin was diluted in 2×Ca²⁺ Bindingbuffer, 2.5% BSA and was added to all wells at a final concentration of3×10⁴ cpm/well. The plates were then sealed, centrifuged at 1000 rpm for15 minutes and placed on orbital shaker for 2 hours. The analysis wasperformed on a 1450 Microbeta Trilux (Wallac). Percent above backgroundrepresents the percentage amount of counts per minute above the negativecontrols. Percents greater than or equal to 30% above background areconsidered positive.

The following PRO polypeptides tested positive in this assay: PRO719.

Example 134 Human Venous Endothelial Cell Calcium Flux Assay (Assay 68)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to stimulate calcium flux in humanumbilical vein endothelial cells (HUVEC, Cell Systems). Calcium influxis a well documented response upon binding of certain ligands to theirreceptors. A test compound that results in a positive response in thepresent calcium influx assay can be said to bind to a specific receptorand activate a biological signaling pathway in human endothelial cells.This could ultimately lead, for example, to endothelial cell division,inhibition of endothelial cell proliferation, endothelial tubeformation, cell migration, apoptosis, etc.

Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) ingrowth media (50:50 without glycine, 1% glutamine, 10 mM Hepes, 10% FBS,10 ng/ml bFGF), were plated on 96-well microtiter ViewPlates-96 (PackardInstrument Company Part #6005182) microtiter plates at a cell density of2×10⁴ cells/well. The day after plating, the cells were washed threetimes with buffer (HBSS plus 10 mM Hepes), leaving 100 μl/well. Then 100μl/well of 8 μM Fluo-3 (2×) was added. The cells were incubated for 1.5hours at 37° C./5% CO₂. After incubation, the cells were then washed 3×with buffer (described above) leaving 100 μl/well. Test samples of thePRO polypeptides were prepared on different 96-well plates at 5×concentration in buffer. The positive control corresponded to 50 μMionomycin (5×); the negative control corresponded to Protein 32. Cellplate and sample plates were run on a FLWPR (Molecular Devices) machine.The FLIPR machine added 25 μl of test sample to the cells, and readingswere taken every second for one minute, then every 3 seconds for thenext three minutes.

The fluorescence change from baseline to the maximum rise of the curve(Δ change) was calculated, and replicates averaged. The rate offluorescence increase was monitored, and only those samples which had aΔ change greater than 1000 and a rise within 60 seconds, were consideredpositive.

The following PRO polypeptides tested positive in the present assay:PRO771.

Example 135 Induction of c-fos in Endothelial Cells (Assay 34)

This assay is designed to determine whether PRO polypeptides show theability to induce c-fos in endothelial cells. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) ingrowth media (50% Ham's F12 w/o GHT: low glucose, and 50% DMEM withoutglycine: with NaHCO3, 1% glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF)were plated on 96-well microtiter plates at a cell density of 1×10⁴cells/well. The day after plating, the cells were starved by removingthe growth media and treating the cells with 100 μl/well test samplesand controls (positive control=growth media; negative control=Protein32buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The cellswere incubated for 30 minutes at 37° C., in 5% CO₂. The samples wereremoved, and the first part of the bDNA kit protocol (ChironDiagnostics, cat. #6005-037) was followed, where each capitalizedreagent/buffer listed below was available from the kit.

Briefly, the amounts of the TM Lysis Buffer and Probes needed for thetests were calculated based on information provided by the manufacturer.The appropriate amounts of thawed Probes were added to the TM LysisBuffer. The Capture Hybridization Buffer was warmed to room temperature.The bDNA strips were set up in the metal strip holders, and 100 μl ofCapture Hybridization Buffer was added to each b-DNA well needed,followed by incubation for at least 30 minutes. The test plates with thecells were removed from the incubator, and the media was gently removedusing the vacuum manifold. 100 μl of Lysis Hybridization Buffer withProbes were quickly pipetted into each well of the microtiter plates.The plates were then incubated at 55° C. for 15 minutes. Upon removalfrom the incubator, the plates were placed on the vortex mixer with themicrotiter adapter head and vortexed on the #2 setting for one minute.80 μl of the lysate was removed and added to the bDNA wells containingthe Capture Hybridization Buffer, and pipetted up and down to mix. Theplates were incubated at 53° C. for at least 16 hours.

On the next day, the second part of the bDNA kit protocol was followed.Specifically, the plates were removed from the incubator and placed onthe bench to cool for 10 minutes. The volumes of additions needed werecalculated based upon information provided by the manufacturer. AnAmplifier Working Solution was prepared by making a 1:100 dilution ofthe Amplifier Concentrate (20 fm/μl) in AL Hybridization Buffer. Thehybridization mixture was removed from the plates and washed twice withWash A. 50 μl of Amplifier Working Solution was added to each well andthe wells were incubated at 53° C. for 30 minutes. The plates were thenremoved from the incubator and allowed to cool for 10 minutes. The LabelProbe Working Solution was prepared by making a 1:100 dilution of LabelConcentrate (40 pmoles/μl) in AL Hybridization Buffer. After the10-minute cool-down period, the amplifier hybridization mixture wasremoved and the plates were washed twice with Wash A. 50 μl of LabelProbe Working Solution was added to each well and the wells wereincubated at 53° C. for 15 minutes. After cooling for 10 minutes, theSubstrate was warmed to room temperature. Upon addition of 3 μl ofSubstrate Enhancer to each ml of Substrate needed for the assay, theplates were allowed to cool for 10 minutes, the label hybridizationmixture was removed, and the plates were washed twice with Wash A andthree times with Wash D. 50 μl of the Substrate Solution with Enhancerwas added to each well. The plates were incubated for 30 minutes at 37°C. and RLU was read in an appropriate luminometer.

The replicates were averaged and the coefficient of variation wasdetermined. The measure of activity of the fold increase over thenegative control (Protein 32/HEPES buffer described above) value wasindicated by chemiluminescence units (RLU). The results are consideredpositive if the PRO polypeptide exhibits at least a two-fold value overthe negative buffer control. Negative control=1.00 RLU at 1.00%dilution. Positive control 8.39 RLU at 1.00% dilution.

The following PRO polypeptides tested positive in this assay: PRO474.

Example 136 Induction of Pancreatic β-Cell Precursor Differentiation(Assay 89)

This assay shows that certain polypeptides of the invention act toinduce differentiation of pancreatic β-cell precursor cells into maturepancreatic β-cells and, therefore, are useful for treating variousinsulin deficient states in mammals, including diabetes mellitus. Theassay is performed as follows. The assay uses a primary culture of mousefetal pancreatic cells and the primary readout is an alteration in theexpression of markers that represent either β-cell precursors or matureβ-cells. Marker expression is measured by real time quantitative PCR(RTQ-PCR); wherein the marker being evaluated is insulin.

The pancreata are dissected from E14 embryos (CD1 mice). The pancreataare then digested with collagenase/dispase in F12/DMEM at 37° C. for 40to 60 minutes (collagenase/dispase, 1.37 mg/ml, Boehringer Mannheim,#1097113). The digestion is then neutralized with an equal volume of 5%BSA and the cells are washed once with RPMI1640. At day 1, the cells areseeded into 12-well tissue culture plates (precated with laminin, 20μg/ml in PBS, Boehringer Mannheim, #124317). Cells from pancreata from1–2 embryos are distributed per well. The culture medium for thisprimary cuture is 14F/1640. At day 2, the media is removed and theattached cells washed with RPMI/1640. Two mls of minimal media are addedin addition to the protein to be tested. At day 4, the media is removedand RNA prepared from the cells and marker expression analyzed by realtime quantitative RT-PCR. A protein is considered to be active in theassay if it increases the expression of the relevant β-cell marker ascompared to untreated controls.

14F/1640 is RPMI1640 (Gibco) plus the following:

group A 1:1000

group B 1:1000

recombinant human insulin 10 μg/ml

Aprotinin (50 μg/ml) 1:2000 (Boehringer manheim #981532)

Bovine pituitary extract (13PE) 60 μg/ml

Gentamycin 100 ng/ml

Group A: (in 10 ml PBS)

Transferrin, 100 mg (Sigma T2252)

Epidermal Growth Factor, 100 μg (BRL 100004)

Triiodothyronine, 10 μl of 5×10³¹ ⁶ M (Sigma T5516)

Ethanolamine, 100 μl of 10⁻¹ M (Sigma E0135)

Phosphoethalamine, 100 μl of 10⁻¹ M (Sigma P0503)

Selenium, 4 μl of 10⁻¹ M (Aesar #12574)

Group C: (in 10 ml 100% ethanol)

Hydrocortisone, 2 μl of 5×10⁻³ M (Sigma #H0135)

Progesterone, 100 μl of 1×10⁻³ M (Sigma #P6149)

Forskolin, 500 μl of 20 mM (Calbiochem #344270)

Minimal media:

RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml), gentamycin(100 ng/ml), aprotinin (50 μg/ml) and BPE (15 μg/ml).

Defined media:

RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml), gentamycin(100 ng/ml) and aprotinin (50 μg/ml).

The following polypeptides were positive in this assay: PRO788 andPRO162.

Example 137 Stimulation of Endothelial Cell Proliferation (Assay 8)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to stimulate adrenal corticalcapillary endothelial cell (ACE) growth. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

Bovine adrenal cortical capillary endothelial (ACE) cells (from primaryculture, maximum of 12–14 passages) were plated in 96-well plates at 500cells/well per 100 microliter. Assay media included low glucose DMEM,10% calf serum, 2 mM glutamine, and 1×penicillin/streptomycin/fungizone. Control wells included the following:(1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus VEGF (5ng/ml); and (4) ACE cells plus FGF (5 ng/ml). The control or testsample, (in 100 microliter volumes), was then added to the wells (atdilutions of 1%, 0.1% and 0.01%, respectively). The cell cultures wereincubated for 6–7 days at 37° C./5% CO₂. After the incubation, the mediain the wells was aspirated, and the cells were washed 1× with PBS. Anacid phosphatase reaction mixture (100 microliter; 0.1M sodium acetate,pH 5.5, 0.1% Triton X-100, 10 mM p-nitrophenyl phosphate) was then addedto each well. After a 2 hour incubation at 37° C., the reaction wasstopped by addition of 10 microliters 1N NaOH. Optical density (OD) wasmeasured on a microplate reader at 405 nm.

The activity of a PRO polypeptide was calculated as the fold increase inproliferation (as determined by the acid phosphatase activity, OD 405um) relative to (1) cell only background, and (2) relative to maximumstimulation by VEGF. VEGF (at 3–10 ng/ml) and FGF (at 1–5 ng/nm) wereemployed as an activity reference for maximum stimulation. Results ofthe assay were considered “positive” if the observed stimulation was≧50% increase over background. VEGF (5 ng/ml) control at 1% dilutiongave 1.24 fold stimulation; FGF (5 ng/ml) control at 1% dilution gave1.46 fold stimulation.

The following PRO polypeptides tested positive in this assay: PRO1075.

Example 138 Mouse Mesengial Cell Inhibition Assay (Assay 114)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to inhibit the proliferation of mousemesengial cells in culture. PRO polypeptides testing positive in thisassay would be expected to be useful for the therapeutic treatment ofsuch diseases or conditions where inhibition of mesengial cellproliferation would be beneficial such as, for example, cystic renaldysplasia, polycystic kidney disease, or other kidney diseaseassoiciated with abnormal mesengial cell proliferation, renal tumors,and the like.

On day 1, mouse mesengial cells are plated on a 96 well plate in growthmedium (a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham'sF12medium, 95%; fetal bovine serum, 5%; supplemented with 14 mM HEPES)and then are allowed to grow overnight. On day 2, the PRO polypeptide isdiluted at 2 different concentrations (1%, 0.1%) in serum-free mediumand is added to the cells. The negative control is growth medium withoutadded PRO polypeptide. After the cells are allowed to incubate for 48hours, 20 μl of the Cell Titer 96 Aqueous one solution reagent (Promega)is added to each well and the colormetric reaction is allowed to proceedfor 2 hours. The absorbance (OD) is then measured at 490 nm. A positivein the assay is an absorbance reading which is at least 10% above thenegative control.

The following PRO polypeptides tested positive in this assay: PRO200 andPRO697.

Example 139 Chondrocyte Proliferation Assay (Assay 111)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to induce the proliferation and/orredifferentiation of chondrocytes in culture. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis.

Porcine chondrocytes are isolated by overnight collagenase digestion ofarticular cartilage of the metacarpophalangeal joint of 4–6 month oldfemale pigs. The isolated cells are then seeded at 25,000 cells/cm² inHam F-12 containing 10% FBS and 4 μg/ml gentamycin. The culture media ischanged every third day and the cells are reseeded to 25,000 cells/cm²every five days. On day 12, the cells are seeded in 96 well plates a5,000 cells/well in 100 μl of the same media without serum and 100 μl ofeither serum-free medium (negative control), staurosporin (finalconcentration of 5 nM; positive control) or the test PRO polypeptide areadded to give a final volume of 200 μl/well. After 5 days at 37° C., 20μl of Alamar blue is added to each well and the plates are incubated foran additional 3 hours at 37° C. The fluorescence is then measured ineach well (Ex: 530 nm; Em: 590 um). The fluorescence of a platecontaining 200 μl of the serum-free medium is measured to obtain thebackground. A positive result in the assay is obtained when thefluorescence of the PRO polypeptide treated sample is more like that ofthe positive control than the negative control.

The following PRO polypeptides tested positive in this assay: PRO181,PRO200 and PRO322.

Example 140 Rat DRG Neuronal Survival Inhibition Assay

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to inhibit the survival of neuralcells in culture. Polypeptides testing positive in this assay areexpected to be useful for the therapeutic treatment of neuropathicconditions which are associated with undesirable neural cellproliferation including, for example, neuroblastomas, gliomas,glioblastomas, and the like.

A heterogeneous population of neural cells freshly isolated from E14 ratembryo dorsal root ganglia are diluted in complete medium and are platedat 5,000 cells/well on polyurethane pretreated plates containing 50 μlF12 complete media. Test PRO polypeptides (50 μl, one concentration)with 50 μl additional assay media are then added to test for survivalinhibition activity. Negative controls are treated with 100 μl ofcomplete medium alone. After 3 days incubation, the cells are stainedwith CMFDA and fixed after 1 hour with 4% paraformaldehyde. Cells arethen quantified by NIH image analysis. A positive in the assay is cellnumbers in the treated well(s) being less than 0.5 of the untreatedcontrol well(s).

The following PRO polypeptides tested positive in this assay: PRO195 andPRO701.

Example 141 Tissue Expression Distribution

Oligonucleotide probes were constructed from some of the PROpolypeptide-encoding nucleotide sequences shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200–600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions with cDNAlibraries isolated from different human adult and/or fetal tissuesources and analyzed by agarose gel electrophoresis so as to obtain aquantitative determination of the level of expression of the PROpolypeptide-encoding nucleic acid in the various tissues tested.Knowledge of the expression pattern or the differential expression ofthe PRO polypeptide-encoding nucleic acid in various different humantissue types provides a diagnostic marker useful for tissue typing, withor without other tissue-specific markers, for determining the primarytissue source of a metastatic tumor, and the like. These assays providedthe following results.

Tissues With Tissues Lacking DNA Molecule Significant ExpressionSignificant Expression DNA40954-1233 liver, lung brain DNA41404-1352lung, kidney liver, retina, pancreas DNA44179-1362 liver lung, brainDNA45234-1277 kidney liver, placenta, brain DNA45415-1318 thyroid,brain, kidney liver, bone marrow DNA45417-1432 thyroid, brain, kidney,liver bone marrow DNA45493-1349 liver, kidney brain DNA48306-1291 brain,kidney pancreas, liver DNA48328-1355 thyroid, brain, liver, bone marrowkidney DNA48329-1290 brain, bone marrow, liver, thyroid kidneyDNA49624-1279 placenta liver, lung, kidney, brain DNA50911-1288 brainplacenta DNA50914-1289 brain, kidney, liver placenta DNA53906-1368 lung,kidney brain DNA53912-1457 lung, liver, kidney, brain pancreasDNA53977-1371 lung, liver, kidney, brain, pancreas bone marrowDNA54002-1367 bone marrow, liver, lung, thyroid, brain kidneyDNA55737-1345 bone marrow, kidney liver, brain DNA57039-1402 pigmentepithelium lung, brain, liver, kidney DNA57253-1382 lung, brain, liver,placenta kidney DNA58747-1384 lung, brain, kidney, pancreas, thyroidliver DNA23318-1211 spleen, brain, heart, cartilage colon tumor,prostate DNA39975-1210 brain, colon tumor, THP-1 macrophages heartDNA39979-1213 dendrocytes, cartilage, spleen, substantia nigra, heartuterus, prostate DNA41386-1316 HUVEC, cartilage, substantia nigra, colontumor, dendrocytes uterus DNA50919-1361 HUVEC, brain, prostate,cartilage, heart, spleen, colon tumor uterus DNA52185-1370 dendrocytessubstantia nigra, hippocampus, uterus DNA42663-1154 uterus, spleen, bonecartilage, HUVEC, colon marrow tumor DNA50980-1286 placenta, adrenalbone marrow, uterus, cartilage gland, prostate

Example 142 In situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1:169–176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedde human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [³³-P] UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10μ; each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase wereadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipettedonto DE81 paper. The remaining solution was loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit was inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE were added. 1 μlof the final product was pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe was run on a TBE/urea gel. 1–3 μl of the probe or 5 μl of RNAMrk III were added to 3 μl of loading buffer. After heating on a 95° C.heat block for three minutes, the gel was immediately placed on ice. Thewells of gel were flushed, the sample loaded, and run at 180–250 voltsfor 45 minutes. The gel was wrapped in saran wrap and exposed to XARfilm with an intensifying screen in −70° C. freezer one hour toovernight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminium trays andthawed at room temperature for 5 minutes. The trays were placed in 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/mn stock in 250 ml prewarmedRNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-Embedded Sections

The slides were deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes)—human embryo, or 8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30minutes)—formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationwere performed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)—saturated filter paper. The tissue was covered with 50 μlof hybridization buffer (3.75 g Dextran Sulfate+6 ml SQ H₂O), vortexedand heated in the microwave for 2 minutes with the cap loosened. Aftercooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC and 9 ml SQ H₂O wereadded, the tissue was vortexed well, and incubated at 42° C. for 1–4hours.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400ml 20×SSC+16 ml 0.25 M EDTA, V_(f)=4 L), followed by RNaseA treatment at37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at roomtemperature. The stringency wash conditions were as follows: 2 hours at55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4 L).

F. Oligonucleotides

In situ analysis was performed on a variety of DNA sequences disclosedherein. The oligonucleotides employed for these analyses were derivedfrom the nucleotide sequences disclosed herein and generally range fromabout 40 to 55 nucleotides in length.

G. Results

In situ analysis was performed on a variety of DNA sequences disclosedherein. The results from these analyses are as follows.

(1) DNA29101-1122 (PRO200)

Fetal:

Lower limb expression in developing lower limb bones at the edge of thecartilagenous anlage (i.e. around the outside edge); in developingtendons, in vascular smooth muscle and in cells embracing developingskeletal muscle myocytes and myotubes. Expression also observed at theepiphyseal growth plate. Lymph node expression in marginal sinus ofdeveloping lymph nodes. Thymus expression in the subcapsular region ofthe thymic cortex, possibly representing either the subcapsularepithelial cells or the proliferating, double negative, thymocytes thatare found in this region. Spleen is negative. Trachea expression insmooth muscle. Brain (cerebral cortex) focal expression in corticalneurones. Spinal cord negative. Small intestine expression in smoothmuscle. Thyroid—generalized expression over thyroid epithelium. Adrenalis negative. Liver expression in ductal plate cells. Stomach expressionin mural smooth muscle. Fetal skin expression in basal layer of squamousepithelium. Placenta expression in interstitial cells in trophoblasticvilli. Cord expression in wall of arteries and vein.

Comments: Expression pattern suggests that PRO200 may be involved incell differentiation/proliferation.

High expression was observed at the following additional sites: Chimpovary—granulosa cells of maturing follicles, lower intensity signalobserved over the cal cells. Chimp parathyroid—high expression overchief cells. Human fetal testis—moderate expression over stromal cellssurrounding developing tubules. Human fetal lung—high expression overchondrocytes in developing bronchial tree, and low level expression overbranching bronchial epithelium. Specific expression was not observedover the renal cell, gastric and colonic carcinomas. Fetal tissuesexamined (E12–E16 weeks) include: placenta, umbilical cord, liver,kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus,stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinalcord, body wall, pelvis and lower limb. Adult tissues examined: liver,kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung,skin, cerebral cortex (rm), hippocampus(rm), cerebellum(rm), penis, eye,bladder, stomach, gastric carcinoma, colon, colonic carcinoma andchondrosarcoma. Acetominophen induced liver injury and hepaticcirrhosis.

(2) DNA30867-1335 (PRO218)

Low level expression over numerous epithelia including fetal smallintestine, fetal thyroid, chimp gastric epithelium. Expression also seenover malignant cells in a renal cell carcinoma. Expression in fetalbrain, over cortex. The distribution does not suggest an obviousfunction. Human fetal tissues examined (E12–E16 weeks) include:placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,heart, great vessels, oesophagus, stomach, small intestine, spleen,thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lowerlimb. Adult human tissues examined: kidney (normal and end-stage),bladder, adrenal, spleen, lymph node, pancreas, lung, skin, eye (inc.retina), colon, bladder, liver (normal, cirrhotic, acute failure),heart, clear cell carcinoma of kidney, gastric adenocarcinoma,colorectal carcinoma. Non-human primate tissues examined: Chimp tissues:salivary gland, stomach, thyroid, parathyroid, tongue, thymus, ovary,lymph node, peripheral nerve. Rhesus Monkey tissues: cerebral cortex,hippocampus, cerebellum, penis.

(3) DNA40021-1154 (PRO285)

Low levels of expression observed in the placenta and over hematopoieticcells in the mouse fetal liver. No expression was detected in eitherhuman fetal, adult or chimp lymph node and no expression was detected inhuman fetal or human adult spleen. Fetal tissues examined (E12–E16weeks) include: placenta, umbilical cord, liver, kidney, adrenals,thyroid, lungs, heart, great vessels, oesophagus, stomach, smallintestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall,pelvis and lower limb. Adult tissues examined: liver, kidney, adrenal,myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cerebralcortex (rm), hippocampus(rm), cerebellum(rm), brain infarct (human),cerebritis (human),penis, eye, bladder, stomach, gastric carcinoma,colon, colonic carcinoma, thyroid (chimp), parathyroid (chimp) ovary(chimp) and chondrosarcoma. Acetominophen induced liver injury andhepatic cirrhosis.

(4) DNA39523-1192 (PRO273)

Expression over epithelium of mouse embryo skin as well as over basalepithelium and dermis of human fetal skin. Basal epithelial pegs of thesquamous mucosa of the chimp tongue are also positive. Expression over asubset of cells in developing glomeruli of fetal kidney, adult renaltubules, and over “thyroidized” epithelium in end-stage renal disease,low expression in a renal cell carcinoma, probably over the epithelialcells. Low level expression over stromal cells in fetal lung. Expressionover stromal cells in the apical portion of gastric glands. Highexpression in the lamina propria of the fetal small intestinal villi,normal colonic mucosa and over stromal cells in a colonic carcinoma.Strong expression over benign connective tissue cells in the hylanizedstroma of a sarcoma. Expression over stromal cells in the placentalvilli and the splenic red pulp. In the brain, expression over corticalneurones. Connective tissue surrounding developing bones and over nervesheath cells in the fetus. Fetal tissues examined (E12–E16 weeks)include: placenta, umbilical cord, liver, kidney, adrenals, thyroid,lungs, heart, great vessels, oesophagus, stomach, small intestine,spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis andlower limb. Adult tissues examined: liver, kidney, adrenal, myocardium,aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm),hippocampus(rm), eye, stomach, gastric carcinoma, colon, coloniccarcinoma, thyroid chimp, parathyroid (chimp) ovary (chimp) andchondrosarcoma. Acetominophen induced liver injury and hepaticcirrhosis.

Expression was present in many cells in the outer layers (and ll) of themonkey cerebral cortex. A small subset of cells in the deeper corticallayers also expressed mRNA for this chemokine homolog. Scattered cellswithin the molecular layers of the hippocampus and bordering the inneredge of the dentate gyrus contained chemokine homolog mRNA. Noexpression was detected within the cerebellar cortex. Chemokine homologexpression is not observed in infarcted brain, where cell death hasoccurred in the regions where the chemokine homolog normally isexpressed. This probe could possibly serve as a marker of a subset ofneurons of outer layers of the cerebral cortex and could possibly revealneuronal migration disorders. Abnormal neuronal migration is a possiblecause of some seizure disorders and schizophrenia. In order to gain abetter appreciation of the distribution of this mRNA we will testwhether the probe will cross-hybridize with mouse brain tissue.

Also shows intriguing and specific patterns of hybridization withinpostnatal day (P)10 and adult mouse brains. In one sagittal section ofP10 mouse brain, strong signal was observed scatered within themolecular layer of the hippocampus and inner edges of the dentate gyrus.Cells in the presubiculum were moderately labeled; the signal extendedin a strong band through outer layers of the retrosplenial cortes to theoccipital cortex, where the signal diminished to background levels. Asmall set of positive neurons were detected in deeper regions of P10motor cortex; neurons in outer layers of P10 cortex did not exhibitsignal above background levels. Moderate hybridization signal was alsodetected in the inferior colliculus. Chemokine homolog signal in theadult mouse brain was evaluated in three coronal sections at differentlevels. Strong signal was detected in the septum and in scatteredneurons in the pontine nuclei and motor root of the trigeminal nerve;moderate signal was seen in the molecular layers of the hippocampus andouter layers of the retrosplenial cortex.

(5) DNA39979-1213 (PRO296)

Widespread expression in fetal in adult tissues. Expressed in a varietyof fetal and adult epithelia, skeletal and cardiac muscle, developing(including retina) and adult CNS, thymic epithelium, placental villi,hepatocytes in cirrhotic and acetaminophen induced toxicity. Highlyexpressed in hypertrophic chondrocytes in developing skeletal system.The overall expression pattern, while not completely overlapping (notexpressed in glomeruli, more widely expressed in CNS), is not disimilarto VEGF. A possible role in angiogenesis should therefore be considered.Human fetal tissues examined (E12–E16 weeks) include: placenta,umbilical cord, liver, kidney, adrenals, thyroid, lungs, great vessels,stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinalcord, body wall, pelvis, testis and lower limb. Adult human tissuesexamined: kidney (normal and end-stage), adrenal, spleen, lymph node,pancreas, lung, eye (inc. retina), bladder, liver (normal, cirrhotic,acute failure). Non-human primate tissues examined: Chimp tissues:adrenal. Rhesus Monkey tissues: cerebral cortex, hippocampus,cerebellum.

(6) DNA52594-1270 (PRO868)

Expression over neuronal cells in fetal dorsal root ganglia, spinalcord, developing enteric neurons, cortical neurons. Low level expressionalso seen in placental trophoblast. In adult tissues the only site wherenotable expression was observed was the normal adult prostate; as suchit may represent a possible prostate cell surface receptor targetantigen. Studies to further characterize the expression in adult tissuesseem warranted. Low level expression also observed in aliposarcoma.Fetal tissues examined (E12–E16 weeks) include: placenta, umbilicalcord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,oesophagus, stomach, small intestine, spleen, thymus, pancreas, brain,eye, spinal cord, body wall, pelvis and lower limb. Adult human tissuesexamined: liver, kidney, adrenal, myocardium, aorta, spleen, lung, skin,chondrosarcoma, eye, stomach, gastric carcinoma, colon, coloniccarcinoma, renal cell carcinoma, prostate, bladder mucosa and gallbladder. Acetominophen induced liver injury and hepatic cirrhosis.Rhesus tissues examined: cerebral cortex (rm), hippocampus(rm),cerebellum. Chimp tissues examined: thyroid, parathyroid, ovary, nerve,tongue, thymus, adrenal, gastric mucosa and salivary gland. WIG-1(WISP-1), WIG-2 (WISP-2) and WIG-5 (WISP-3) expression in human breastcarcinoma and normal breast tissue, Wig-2 in lung carcinoma, and Wig-5in colon carcinoma.

(7) DNA64907-1163 (PRO1330)

In human fetal tissues there was strong specific expression overartrerial, venous, capillary and sinusoidal endothelium in all tissuesexamined, except for fetal brain. In normal adult tissues expression waslow to absent, but when present appeared expression was confined to thevasculature. Highest expression in adult tissues was observed regionallyin vessels running within the white matter of rhesus brain—thesignificance of this pattern is unclear. Elevated expression observed invasculature of many inflamed and diseased tissues, including tumorvasculature. In some of these tissues it was unclear if expression wassoley confined to vascular endothelium. In the 15 lung tumors examinedno expression was seen over the malignant epithelium, however, vascularexpression was observed in many of the tumors and adjacent lung tissue.Moderate, apparently non-specific background, was seen with this probeover hyalinised collagen and sites of tissue necrosis. In the abscenceof a sense control, however, it is not possible to be absolutely certainthat all of this signal is non-specific. Some signal, also thought to benon-specific, was seen over the chimp gastric mucosa, transitional cellepithelium of human adult bladder and fetal retina.

(8) DNA49624-1279 (PRO545)

Expression of the ADAM family molecule, ADAM 12 (DNA49624-1279) observedin normal human lung, lung tumor, normal colon and colon carcinoma.

(9) DNA59294-1381 (PRO1031)

The expression of this IL17 homologue was evaluated in a panelconsisting of normal adult and fetal tissues and tissues withinflammation, predominantly chronic lymphocytic inflammation. This panelis designed to specifically evaluate the expression pattern in immunemediated inflammatory disease of novel proteins that modulate Tlymphocyte function (stimulatory or inhibitory). This protein whenexpressed as an Ig-fusion protein was immunostimulatory in a dosedependent fashion in the human mixed lymphocyte reaction (MLR); itcaused a 285% and 147% increase above the baseline stimulation indexwhen utilized at two different concentrations (1.0% and 0.1% of a 560 nMstock). Summary: expression was restricted to muscle, certain types ofsmooth muscle in the adult and in skeletal and smooth muscle in thehuman fetus. Expression in adult human was in smooth muscle of tubularorgans evaluated including colon and gall bladder. There no expressionin the smooth muscle of vessels or bronchi. No adult human skeletalmuscle was evaluated. In fetal tissues there was moderate to highdiffuse expression in skeletal muscle the axial skeleton and limbs.There was weak expression in the smooth muscle of the intestinal wallbut no expression in cardiac muscle. Adult human tissues withexpression: Colon. there was low level diffuse expression in the smoothmuscle (tunica muscularis) in 5 specimens with chronic inflammatorybowel disease. Gall bladder: there was weak to low level expression inthe smooth muscle of the gall bladder. Fetal human tissues withexpression: there was moderate diffuse expression in skeletal muscle andweak tolow expression in smooth muscle; there was no expression in fetalheart or any other fetal organ including liver, spleen, CNS, kidney,gut, lung. Human tissues with no expression: lung with chronicgranulomatous inflammation and chronic bronchitis (5 patients),peripheral nerve, prostate, heart, placenta, liver (disease multiblock),brain (cerbrum and cerebellum), tonsil (reactive hyperplasia),peripheral lymph node, thymus.

(10) DNA45416–1251 (PRO362)

The expression of this novel protein was evaluated in a variety of humanand non-human primate tissues and was found to be highly restricted.Expression was present only in alveolar macrophages in the lung and inKupffer cells of the hepatic sinusoids. Expression in these cells wassignificantly increased when these distinct cell populations wereactivated. Though these two subpopulations of tissue macrophages arelocated in different organs, they have similar biological functions.Both types of these phagocytes act as biological filters to removematerial from the blood stream or airways including pathogens, senescentcells and proteins and both are capable of secreting a wide variety ofimportant proinflammatory cytokines. In inflamed lung (7 patientsamples) expression was prominent in reactive alveolar macrophage cellpopulations defined as large, pale often vacuolated cells present singlyor in aggregates within alveoli and was weak to negative in normal,non-reactive macrophages (single scattered cells of normal size).Expression in alveolar macrophages was increased during inflammationwhen these cells were both increased in numbers and size (activated).Despite the presence of histocytes in areas of interstial inflammtionand peribronchial lymphoid hyperplasia in these tissues, expression wasrestricted to alveolar macrophages. Many of the inflamed lungs also hadsome degree of suppurative inflammation; expression was not present inneutrophilic granulocytes. In liver, there was strong expression inreactive/activated Kupffer cells in livers with acute centrilobularnecrosis (acetominophen toxicity) or fairly marked periportalinflammtion. However there was weak or no expression in Kupffer cells innormal liver or in liver with only mild inflammation or mild to moderatelobular hyperplasia/hypertrophy. Thus, as in the lung, there wasincreased expression in activated/reactive cells. There was noexpression of this molecule in histiocytes/macropahges present ininflamed bowel, hyperplastic/reactive tonsil or normal lymph node. Thelack of expression in these tissues which all contained histiocyticinflammation or resident macrophage populations strongly supportsrestricted expression to the unique macrophage subset populationsdefined as alveolar macrophage and hepatic Kupffer cells. Spleen or bonemarrow was not available for evaluation. Human tissues evaluated whichhad no detectable expression included: Inflammatory bowel disease (7patient samples with moderate to severe disease), tonsil with reactivehyperplasia, peripheral lymph node, psoriatic skin (2 patient sampleswith mild to moderate disease), heart, peripheral nerve. Chimp tissuesevaluated which had no detectable expression included: tongue, stomach,thymus.

(11) DNA52196-1348 (PRO733)

Generalized low level signal in many tissues and in many cell types.While endothelial cell expression was observed it was not a prominentfeature in either fetal, normal or diseased tissues. Human tissues:moderate expression over fetal liver (mainly hepatocytes), lung, skin,adrenal and heart. Fetal spleen, small intestine, brain and eye arenegative. Adult normal kidney, bladder epithelium, lung, adrenal,pancreas, skin—all negative. Expression in adult human liver (normal anddiseased), renal tubules in end-stage renal disease, adipose tissue,sarcoma, colon, renal cell carcinoma, hepatocellular carcinoma, squamouscell carcinoma. Non human primate tissues: chimp salivary gland,vessels, stomach, tongue, peripheral nerve, thymus, lymph node, thyroidand parathyroid. Rhesus spinal cord negative, cortical and hippocampalneurones positive.

Example 143 Isolation of cDNA Clones Encoding a Human PRO4993

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA85042. In some cases, the DNA85042 consensussequence derives from an intermediate consensus DNA sequence which wasextended using repeated cycles of BLAST and phrap to extend thatintermediate consensus sequence as far as possible using the sources ofEST sequences discussed above. Based on the DNA85042 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO4993.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer 5′-AGATGTGAAGGTGCAGGTGTGCCG-3′ (SEQ ID NO:619)-   reverse PCR primer 5′-GAACATCAGCGCTCCCGGTAATTCC-3′ (SEQ ID NO:620)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA85042 sequence which had the    following nucleotide sequence    hybridization probe-   5′-CCAGCCTTTGAATGGTACAAAGGAGAGAAGAAGCTCTTCAATGGCC-3′ (SEQ ID NO:621)

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO4993 polypeptide(designated herein as DNA94832-2659 [FIG. 229, SEQ ID NO:611]) and thederived protein sequence for that PRO4993 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 305–307 and a stop signal at nucleotide positions 1361–1363(FIG. 229, SEQ ID NO:611). The predicted polypeptide precursor is 352amino acids long, has a calculated molecular weight of approximately38,429 daltons and an estimated pI of approximately 6.84. Analysis ofthe full-length PRO4993 sequence shown in FIG. 230 (SEQ ID NO:612)evidences the presence of a variety of important polypeptide domains asshown in FIG. 230, wherein the locations given for those importantpolypeptide domains are approximate as described above. CloneDNA94832-2659 has been deposited with ATCC on Jun. 15, 1999 and isassigned ATCC deposit no. 240-PTA.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 230 (SEQ ID NO:612), evidenced sequence identity betweenthe PRO4993 amino acid sequence and the following Dayhoff sequences:P_W05152; LAMP_HUMAN; P_W05157; P_W05155; I56551; OPCM_RAT; AMAL_DROME;DMU78177_(—)1; I₃₇₂₄₆; and NCA1_HUMAN.

Example 144 Isolation of cDNA Clones Encoding Human PRO1559, PRO725 andPRO739

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above. Based upon an observed homology betweenthis consensus sequence and an EST sequence contained within Incyte ESTclone No. 4242090, Incyte EST clone No. 4242090 was purchased and itsinsert was obtained and sequenced. It was discovered that the insertsequence encoded a full-length protein designated herein as PRO1559(FIG. 232; SEQ ID NO:614). The DNA sequence of the insert (DNA68886) isshown in FIG. 231 (SEQ ID NO:613).

A cDNA sequence isolated in the amylase screen described in Example 2above is herein designated DNA43301. The DNA43301 sequence was thencompared to a variety of expressed sequence tag (EST) databases whichincluded public EST databases (e.g., GenBank) and a proprietary EST DNAdatabase (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, Calif.) toidentify existing homologies. The homology search was performed usingthe computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460–480 (1996)). Those comparisons resulting in a BLASTscore of 70 (or in some cases 90) or greater that did not encode knownproteins were clustered and assembled into consensus DNA sequences withthe program “phrap” Phil Green, University of Washington, Seattle,Wash.). The consensus sequence obtained therefrom is herein designatedDNA45458. Based on the DNA45458 consensus sequence, oligonucleotideprobes were generated and used to screen a human fetal brain (LIB153)library prepared as described in paragraph 1 of Example 2 above. Thecloning vector was pRK5B (pRK5B is a precursor of pRK5D that does notcontain the SfiI site; see, Holmes et al., Science, 253:1278–1280(1991)), and the cDNA size cut was less than 2800 bp.

PCR primers (forward and reverse) were synthesized:

-   forward PCR primer (45458.f1) 5′-CCAAACTCACCCAGTGAGTGTGAGC-3′ (SEQ    ID NO:619)-   reverse PCR primer (45458.r1) 5′-TGGGAAATCAGGAATGGTGTTCTCC-3′ (SEQ    ID NO:620)    Additionally, a synthetic oligonucleotide hybridization probe was    constructed from the consensus DNA45458 sequence which had the    following nucleotide sequence    hybridization probe (45458.p1)-   5′-CTTGTTTTCACCATTGGGCTAACTTTGCTGCTAGGAGTTCAAGCCATGCC-3′ (SEQ ID    NO:621)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO725 gene using the probe oligonucleotideand one of the PCR primers.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 161–163 and ending at the stop codon found at nucleotidepositions 455–457 (FIG. 233; SEQ ID NO:615). The predicted polypeptideprecursor is 98 amino acids long, has a calculated molecular weight ofapproximately 11,081 daltons and an estimated pI of approximately 6.68.Analysis of the full-length PRO725 sequence shown in FIG. 234 (SEQ IDNO:616) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 20, a potential N-glycosylationsite from about amino acid 72 to about amino acid 75 and a tyrosinekinase phosphorylation site from about amino acid 63 to about amino acid70. Clone DNA52758-1399 has been deposited with ATCC on Apr. 14, 1998and is assigned ATCC deposit no. 209773.

Analysis of the amino acid sequence of the full-length PRO725polypeptide suggests that it possesses no significant sequencesimilarity to any known protein. However, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced some degree of homologybetween the PRO725 amino acid sequence and the following Dayhoffsequences, POL_BLVAU, PSSP_RAT, CELC36C5_(—)7, AF019234_(—)1, I₄₈₈₆₂,P_R12498, P_P10125, P_R26861, A64527 and P_W20495.

DNA52756, as shown in FIG. 235 (SEQ ID NO:617) and which encodes nativePRO739 polypeptide (FIG. 236; SEQ ID NO:618) was obtained from GenBank.

Example 145 Identification of Receptor/Ligand Interactions

In this assay, various PRO polypeptides are tested for ability to bindto a panel of potential receptor molecules for the purpose ofidentifying receptor/ligand interactions. The identification of a ligandfor a known receptor, a receptor for a known ligand or a novelreceptor/ligand pair is useful for a variety of indications including,for example, targeting bioactive molecules (linked to the ligand orreceptor) to a cell known to express the receptor or ligand, use of thereceptor or ligand as a reagent to detect the presence of the ligand orreceptor in a composition suspected of containing the same, wherein thecomposition may comprise cells suspected of expressing the ligand orreceptor, modulating the growth of or another biological orimmunological activity of a cell known to express or respond to thereceptor or ligand, modulating the immune response of cells or towardcells that express the receptor or ligand, allowing the preparation ofagonists, antagonists and/or antibodies directed against the receptor orligand which will modulate the growth of or a biological orimmunological activity of a cell expressing the receptor or ligand, andvarious other indications which will be readily apparent to theordinarily skilled artisan.

The assay is performed as follows. A PRO polypeptide of the presentinvention suspected of being a ligand for a receptor is expressed as afusion protein comaning the Fc domain of human IgG (an immunoadhesin).Receptor-ligand binding is detected by allowing interaction of theimmunoadhesin polypeptide with cells (e.g. Cos cells) expressingcandidate PRO polypeptide receptors and visualization of boundimmunoadhesin with fluorescent reagents directed toward the Fc fusiondomain and examination by microscope. Cells expressing candidatereceptors are produced by transient transfection, in parallel, ofdefined subsets of a library of cDNA expression vectors encoding PROpolypeptides that may function as receptor molecules. Cells are thenincubated for 1 hour in the presence of the PRO polypeptideimmunoadhesin being tested for possible receptor binding. The cells arethen washed and fixed with paraformaldehyde. The cells are thenincubated with fluorescent conjugated antibody directed against the Fcportion of the PRO polypeptide immunoadhesin (e.g. FITC conjugated goatanti-human-Fc antibody). The cells are then washed again and examined bymicroscope. A positive interaction is judged by the presence offluorescent labeling of cells transfected with cDNA encoding aparticular PRO polypeptide receptor or pool of receptors and an absenceof similar fluorescent labeling of similarly prepared cells that havebeen transfected with other cDNA or pools of cDNA. If a defined pool ofcDNA expression vectors is judged to be positive for interaction with aPRO polypeptide immunoadhesin, the individual cDNA species that comprisethe pool are tested individually (the pool is “broken down”) todetermine the specific cDNA that encodes a receptor able to interactwith the PRO polypeptide immunoadhesin.

In another embodiment of this assay, an epitope-tagged potential ligandPRO polypeptide (e.g. 8 histidine “His” tag) is allowed to interact witha panel of potential receptor PRO polypeptide molecules that have beenexpressed as fusions with the Fc domain of human IgG (immunoadhesins).Following a 1 hour co-incubation with the epitope tagged PROpolypeptide, the candidate receptors are each immunoprecipitated withprotein A beads and the beads are washed. Potential ligand interactionis determined by western blot analysis of the immunoprecipitatedcomplexes with antibody directed towards the epitope tag. An interactionis judged to occur if a band of the anticipated molecular weight of theepitope tagged protein is observed in the western blot analysis with acandidate receptor, but is not observed to occur with the other membersof the panel of potential receptors.

Using these assays, the following receptor/ligand interactions have beenherein identified: PRO337 binds to PRO4993, PRO1559 binds to PRO725,PRO1559 binds to PRO700 and PRO1559 binds to PRO739.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA39987-1184 ATCC 209786 Apr. 21,1998 DNA40625-1189 ATCC 209788 Apr. 21, 1998 DNA23318-1211 ATCC 209787Apr. 21, 1998 DNA39979-1213 ATCC 209789 Apr. 21, 1998 DNA40594-1233 ATCC209617 Feb. 5, 1998 DNA45416-1251 ATCC 209620 Feb. 5, 1998 DNA45419-1252ATCC 209616 Feb. 5, 1998 DNA52594-1270 ATCC 209679 Mar. 17, 1998DNA45234-1277 ATCC 209654 Mar. 5, 1998 DNA49624-1279 ATCC 209655 Mar. 5,1998 DNA48309-1280 ATCC 209656 Mar. 5, 1998 DNA46776-1284 ATCC 209721Mar. 31, 1998 DNA50980-1286 ATCC 209717 Mar. 31, 1998 DNA50913-1287 ATCC209716 Mar. 31, 1998 DNA50914-1289 ATCC 209722 Mar. 31, 1998DNA48296-1292 ATCC 209668 Mar. 11, 1998 DNA32284-1307 ATCC 209670 Mar.11, 1998 DNA36343-1310 ATCC 209718 Mar. 31, 1998 DNA40571-1315 ATCC209784 Apr. 21, 1998 DNA41386-1316 ATCC 209703 Mar. 26, 1998DNA44194-1317 ATCC 209808 Apr. 28, 1998 DNA45415-1318 ATCC 209810 Apr.28, 1998 DNA44189-1322 ATCC 209699 Mar. 26, 1998 DNA48304-1323 ATCC209811 Apr. 28, 1998 DNA49152-1324 ATCC 209813 Apr. 28, 1998DNA49646-1327 ATCC 209705 Mar. 26, 1998 DNA49631-1328 ATCC 209806 Apr.28, 1998 DNA49645-1347 ATCC 209809 Apr. 28, 1998 DNA45493-1349 ATCC209805 Apr. 28, 1998 DNA48227-1350 ATCC 209812 Apr. 28, 1998DNA41404-1352 ATCC 209844 May 6, 1998 DNA44196-1353 ATCC 209847 May 6,1998 DNA52187-1354 ATCC 209845 May 6, 1998 DNA48328-1355 ATCC 209843 May6, 1998 DNA56352-1358 ATCC 209846 May 6, 1998 DNA53971-1359 ATCC 209750Apr. 7, 1998 DNA50919-1361 ATCC 209848 May 6, 1998 DNA44179-1362 ATCC209851 May 6, 1998 DNA54002-1367 ATCC 209754 Apr. 7, 1998 DNA53906-1368ATCC 209747 Apr. 7, 1998 DNA52185-1370 ATCC 209861 May 14, 1998DNA53977-1371 ATCC 209862 May 14, 1998 DNA57253-1382 ATCC 209867 May 14,1998 DNA58847-1383 ATCC 209879 May 20, 1998 DNA58747-1384 ATCC 209868May 14, 1998 DNA57689-1385 ATCC 209869 May 14, 1998 DNA23330-1390 ATCC209775 Apr. 14, 1998 DNA26847-1395 ATCC 209772 Apr. 14, 1998DNA53974-1401 ATCC 209774 Apr. 14, 1998 DNA57039-1402 ATCC 209777 Apr.14, 1998 DNA57033-1403 ATCC 209905 May 27, 1998 DNA34353-1428 ATCC209855 May 12, 1998 DNA45417-1432 ATCC 209910 May 27, 1998 DNA39523-1192ATCC 209424 Oct. 31, 1997 DNA44205-1285 ATCC 209720 Mar. 31, 1998DNA50911-1288 ATCC 209714 Mar. 31, 1998 DNA48329-1290 ATCC 209785 Apr.21, 1998 DNA48306-1291 ATCC 209911 May 27, 1998 DNA48336-1309 ATCC209669 Mar. 11, 1998 DNA44184-1319 ATCC 209704 Mar. 26, 1998DNA48314-1320 ATCC 209702 Mar. 26, 1998 DNA48333-1321 ATCC 209701 Mar.26, 1998 DNA50920-1325 ATCC 209700 Mar. 26, 1998 DNA50988-1326 ATCC209814 Apr. 28, 1998 DNA48331-1329 ATCC 209715 Mar. 31, 1998DNA30867-1335 ATCC 209807 Apr. 28, 1998 DNA55737-1345 ATCC 209753 Apr.7, 1998 DNA49829-1346 ATCC 209749 Apr. 7, 1998 DNA52196-1348 ATCC 209748Apr. 7, 1998 DNA56965-1356 ATCC 209842 May 6, 1998 DNA56405-1357 ATCC209849 May 6, 1998 DNA57530-1375 ATCC 209880 May 20, 1998 DNA56439-1376ATCC 209864 May 14, 1998 DNA56409-1377 ATCC 209882 May 20, 1998DNA56112-1379 ATCC 209883 May 20, 1998 DNA56045-1380 ATCC 209865 May 14,1998 DNA59294-1381 ATCC 209866 May 14, 1998 DNA56433-1406 ATCC 209857May 12, 1998 DNA53912-1457 ATCC 209870 May 14, 1998 DNA50921-1458 ATCC209859 May 12, 1998 DNA29101-1122 ATCC 209653 Mar. 5, 1998 DNA40021-1154ATCC 209389 Oct. 17, 1997 DNA42663-1154 ATCC 209386 Oct. 17, 1997DNA30943-1-1163-1 ATCC 209791 Apr. 21, 1998 DNA64907-1163-1 ATCC 203242Sep. 9, 1998 DNA64908-1163-1 ATCC 203243 Sep. 9, 1998 DNA39975-1210 ATCC209783 Apr. 21, 1998 DNA43316-1237 ATCC 209487 Nov. 21, 1997DNA55800-1263 ATCC 209680 Mar. 17, 1998 DNA94832-2659 240-PTA Jun. 15,1999 DNA52758-1399 ATCC 209773 Apr. 14, 1998

These deposit were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and for at least five (5) years afterthe most recent request for the furnishing of a sample of the depositreceived by the depository. The deposits will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures that all restrictionsimposed by the depositor on the availability to the public of thedeposited material will be irrevocably removed upon the granting of thepertinent U.S. patent. assures permanent and unrestricted availabilityof the progeny of the culture of the deposit to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedhereinwill become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An antibody that specifically binds to the polypeptide of SEQ IDNO:231.
 2. The antibody of claim 1 which is a monoclanal antibody. 3.The antibody of claim 1 which is a humanized antibody.
 4. An antigenbinding fragment of the antibody of claim
 1. 5. The antibody of claim 1which is labeled.